Method of treatment by administering an antibody to human interleukin-5 receptor α chain

ABSTRACT

The present invention provides monoclonal antibodies and humanized antibodies which react specifically with a human interleukin-5 receptor α chain. The invention also provides hybridomas and transformants which produce the antibodies, the monoclonal antibodies and humanized antibodies, a method for detecting an interleukin-5 receptor α chain immunologically by means of these antibodies, as well as a method for diagnosing and treating diseases such as chronic bronchial asthma by means of the monoclonal antibodies and humanized antibodies. The present invention is useful for diagnosis or treatment of diseases such as chronic bronchial asthma.

This application is a division of U.S. patent application No. 09/434,122filed Nov. 5, 1999 now U.S. Pat. No. 6,538,111 currently allowed, whichin turn is a divisional of U.S. patent application No. 08/836,561 filedon May 9, 1997 now U.S. Pat. No. 6,018,032, as a national stage ofInternational Application No.: PCT/JP96/02588 filed Sep. 11, 1996, whichand claims priority to Japanese Application 7-232384 filed Sep. 11,1995. Application Ser. No. 08/836,561 issued as U.S. Pat. No. 6,018,032.

FIELD OF THE INVENTION

The present invention relates to monoclonal antibodies and humanizedantibodies which bind specifically to a human interleukin-5 receptor αchain and which are therefore useful for diagnosis or treatment ofdiseases such as chronic bronchial asthma. The invention also relates tohybridomas and transformants which produce the antibodies, a method fordetecting an interleukin-5 receptor α chain immunologically by means ofthe monoclonal antibodies and humanized antibodies, as well as a methodfor diagnosing and treating diseases such as chronic bronchial asthma bymeans of the monoclonal antibodies and humanized antibodies.

BACKGROUND OF THE INVENTION

Interleukin-5 (hereinafter referred to a “IL-5”) is a kind of lymphokinewhich is secreted by T cells, mast cells and other cells. Murine IL-5 isknown to act as a differentiation and growth factor for B cells andeosinophils. Human IL-5 is known to act mainly as a differentiation andgrowth factor for eosinophils (Advances in Immunology, 57, 145 (1994);Blood, 79, 3101 (1992)). IL-5 exhibits its action through a specificreceptor (IL-5 receptor) which is expressed on the surface of a cellsuch as eosinophil. It has been shown that human and murine IL-5receptors (hereinafter referred to as “IL-5Rs”) are both composed of twodifferent kinds of proteins, an α chain (hereinafter referred to as“IL-5R α”) and a β chain (hereinafter referred to as “IL-5R β”). Inaddition, it is known that the binding of IL-5 to IL-5R is via IL-5R αand that IL-5R β alone can not bind to IL-5 (EMBO J., 9, 4367 (1990);ibid., 10, 2833 (1991); J. Exp. Med., 177, 1523 (1993); ibid., 175, 341(1992); Cell, 66, 1175 (1991), Proc. Natl. Acad. Sci., 89, 7041 (1992)).Furthermore, IL-5R β is known to be a component of receptors forinterleukin-3 (hereinafter referred to as “IL-3”), granulocytemacrophage colony-stimulating factor and others (hereinafter referred toas “GM-CSF”) (Proc. Natl. Acad. Sci., 87, 9655 (1990); Cell, 66, 1165(1991)).

Eosinophils are known to increase in allergic diseases represented bychronic bronchial asthma. Significant infiltration of eosinophils isobserved in airways of a patient with chronic bronchial asthma.Eosinophil contains a cytotoxic granular proteins whose deposit isobserved in airway tissues of a patient with chronic bronchial asthma orat lesion sites of a patient with atopic dermatitis. These facts suggestthat eosinophil plays an important role in the pathogenesis of allergicdisorders such as chronic bronchial asthma, atopic dermatitis and thelike (Adv. Immunol., 39, 177 (1986); Immunol. Today, 13, 501 (1992)).Hence, studying the kinetics of eosinophils is useful for clinicaldiagnosis. On the other hand, human IL-5 acts specifically oneosinophils, so IL-5R is believed to be expressed specifically ineosinophils and can therefore be used as a marker specific to humaneosinophils. Furthermore, IL-5β is a receptor for cytokines such asIL-3, GM-CSF and others, so IL-5R α is believed to be a marker specificto eosinophils. Hence, eosinophils can be detected specifically byimmunocyte staining using an anti-human IL-5R α chain antibody(hereinafter referred to as “anti-hIL-5Rα antibody”). However, noanti-hIL-5R α antibody is presently known that is capable of specificdetection of eosinophils.

Significant cosinophilia was observed in IL-5 transgenic mice (J. Exp.Med., 172, 1425 (1990); ibid. 173, 429 (1991); Int. Immunol., 2, 965(1990)). Eosinophil infiltration in tissues was suppressed by theadministration of an anti-IL-5 antibody in animal models of asthma (Am.Rev. Resir. 147, 548 (1993); ibid., 148, 1623 (1993)). These phenomenaindicate that IL-5 actually plays an important role in eosinophilia andthe infiltration of eosinophils in vivo. It is also reported that IL-5is expressed in airway mucosal tissues of a human patient with chronicbronchial asthma and at lesion sites of a patient with atopic dermatitis(J. Clin. Invest., 87, 1541 (1991); J. Exp. Med., 173, 775 (1991)).Further investigations demonstrate that IL-5 exhibits in vitroviability-enhancing action on human eosinophils (J. Immunol., 143, 2311(1989)) and that IL-5 is an eosinophil-selective activator (J. Exp.Med., 167, 219 (1988)).

Hence, antibodies that bind to IL-5R and which can inhibit thebiological activity of IL-5 are expected to inhibit the activity ofeosinophil, thus being useful in the treatment of allergic diseases suchas chronic bronchial asthma. Anti-mouse IL-5R α antibodies which caninhibit the biological activity of IL-5 were produced by using as anantigen those IL-5-dependent cells which express a large number ofmurine IL-5R on their surfaces (Kokai (Japanese published unexaminedpatent application) No. 108497/91; Int. Immunol., 2, 181 (1990)).However, in the case of humans, no cells are known which express a largenumber of IL-5R and the expression of IL-5R is reported to be very lowin eosinophils (Cell. Immunol., 133, 484 (1991)). Hence, anti-humanIL-5R α antibodies having comparable functions to anti-mouse IL-5R αantibodies are difficult to produce by methods similar to those forproducing the latter. An antibody designated as “α16” is disclosed as anantibody against human IL-5R α in EMBO J., 14, 3395 (1995) but thisantibody does not have any neutralization activity for IL-5R α.

Human IL-5R α gene was obtained by preparing a cDNA library from humaneosinophil (J. Exp. Med., 175, 341 (1992)) or a human promyelocytic cellHL-60 (Cell, 66, 1175 (1991); Kokai No. 78772/94) and screening thelibrary using as a probe an oligo DNA which had been synthesized on thebasis of cDNA of murine IL-5R α or a partial amino acid sequence ofmurine IL-5R α (Kokai No. 54690/94, EMBO J., 9, 4367 (1990)). Thetransfer of the cDNA into a host cell resulted in the creation of a cellhaving hIL-5R α expressed on its surface but the expression level ofhIL-5R in this cell was very low (≦10⁴ molecules) (J. Exp. Med., 177,1523 (1993)). Hence, if one attempts to produce anti-hIL-5R α antibodiesby using this cell as an immunogen, he will find that the relativeamount of hIL-5R α is very small, compared with those of proteins from ahost cell and that the absolute protein amount of hIL-5R α is also verysmall. In addition, approximately 80% homology at an amino acid level isobserved between murine IL-5R α and human IL-5R α and murine IL-5 canbind to human IL-5R with high affinity (J. Exp. Med.,175, 341 (1992)).These facts suggest that human IL-5R α has a lower immunogenicity formice or rats which are commonly used as animals to be immunized. Infact, almost all of our attempts to prepare anti-hIL-5R α antibodiesusing hIL-5R α-expressing cells as an immunogen resulted in a failure.

In the cloning of IL-5R cDNA from a cDNA library of human eosinophil,cDNA encoding soluble human IL-5R α (hereinafter referred to as “shIL-5Rα”) has been obtained which corresponds to the N-terminal amino acidsequence (1-313) of IL-5R α which is defective in the transmembraneregion and onwards (J. Exp. Med.,175, 341 (1992)). When shIL-5R α isused as an immunogen to produce an anti-hIL-5R α antibody, the shIL-5R αshould have the same three dimensional conformation as that of IL-5R αexpressed on the cell surface and it should be one secreted and producedby a eukaryotic host cell in order to obtain an anti-hIL-5R α antibodywhich can inhibit the biological activity of IL-5. In addition, it hasbeen found that the production efficiency of a protein variessignificantly depending on the signal peptide (Protein, Nucleic Acid andEnzyme, 35, 2584 (1990)), so it is necessary to select an appropriatesignal peptide for secretion and production of the protein.

As mentioned above, it has been found that mRNA which is believed toencode only shIL-5R α is expressed in eosinophils. It has been confirmedthat murine IL-5R is expressed not only in eosinophils but also in Bcells and that mRNA which is believed to encode only an extracellularregion of IL-5R α (hereinafter referred to as “smIL-5R α”) is expressedin those cells as well as in the case of humans. In addition, it hasbeen reported that smIL-5R α was detected in blood of mice transplantedwith IL-5R expressing murine chronic B cell leukemia cell line (BCL1) ormodel mice of human autoimmune diseases (J. Immunol. Method, 167, 289(1994)). These suggest the possibility that the increase in the numberof IL-5R expressing cells and their activation may be reflected in theamount of smIL-5R α secreted in blood. Human IL-5R is believed to beexpressed in eosinophils in a limited amount and the increase in thenumber of eosinophils and their activation may be potentially reflectedin the amount of shIL-5R α in blood. Hence, the quantitativedetermination of shIL-5R α is expected to be useful in clinicaldiagnosis.

Any isolated monoclonal antibody which binds specifically to human IL-5Rα is believed to be useful in the diagnosis and treatment of allergicdiseases. However, it should be noted that if a non-human animal-derivedmonoclonal antibody is administered to a human, it is generallyrecognized as a foreign matter such that an antibody against thenon-human animal-derived monoclonal antibody is produced in the humanbody, a reaction with the administered non-human animal-derivedmonoclonal antibody occurs to cause a side effect (J. Clin. Oncol., 2,881 (1984); Blood, 65, 1349 (1985); J. Natl. Cancer Inst., 80, 932(1988); Proc. Natl. Acad. Sci., 82, 1242 (1985)), premature clearance ofthe non-human animal-derived monoclonal antibody occurs (J. Nucl.Med.,26, 1011 (1985); Blood, 65, 1349 (1985); J. Natl. Cancer Inst., 80,937 (1988)), or therapeutic effect of the monoclonal antibody is reduced(J. Immunol., 135, 1530 (1985); Cancer Res., 46, 6489 (1986)).

In order to solve these problems, attempts have been made to convertnon-human animal-derived monoclonal antibodies to human chimericantibodies or human CDR-grafted antibodies (reconstituted humanantibodies) by gene recombinant techniques. A human chimeric antibody isan antibody of which the variable region (hereinafter referred to as “Vregion”) is derived from a non-human animal antibody and the constantregion (hereinafter referred to as “C region”) is derived from a humanantibody (Proc. Natl. Acad. Sci., 81, 6851 (1984)). It has been reportedthat when a human chimeric antibody is administered to a human,antibodies are hardly produced against the non-human animal-derivedmonoclonal antibody and a half-life in blood is increased by a factor of6 (Proc. Natl. Acad. Sci., 86, 4220 (1989)). A human CDR-graftedantibody is a human antibody of which the CDR (complementaritydetermining region) is replaced with the CDR of a non-humananimal-derived antibody (Nature, 321, 522 (1986)). It has been reportedwith experiments on monkeys that a human CDR-grafted antibody has alower immunogenicity, with the half-life in blood being increased by afactor of 4–5 compared with a mouse antibody (J. Immunol., 147, 1352(1991)). However, there is no report about a humanized antibody againsthIL-5R α.

When a humanized antibody which binds specifically to human IL-5R α isadministered to a human, it is expected to cause no production of anantibody against a non-human animal-derived monoclonal antibody, therebyreducing the side effect and prolonging the half-life in blood, whicheventually leads to a high therapeutic effect against allergic diseasessuch as chronic bronchial asthma, atopic dermatitis and the like.

As a result of the recent progresses in protein and genetic engineering,smaller antibody molecules such as single chain antibodies (Science,242, 423 (1988)) and disulfide stabilized antibodies (MolecularImmunology, 32, 249 (1995)) are being prepared. Since single chainantibodies and disulfide stabilized antibodies have smaller molecularweights than monoclonal antibodies and humanized antibodies, they areeffective in transition into tissues and clearance from blood and theirapplication to the imaging technology and the preparation of complexeswith toxins are being underway to provide some promise in therapeuticefficacy (Cancer Research, 55, 318 (1995)). If a single chain antibodyor a disulfide stabilized antibody which binds specifically to a humanIL-5R α chain is produced, high diagnostic and therapeutic effectsagainst allergic diseases and the like are anticipated. However, thereis no report about a single chain antibody and a disulfide stabilizedantibody against a human IL-5R α chain.

SUMMARY OF THE INVENTION

The inventors found that antibodies to a hIL-5R α chain which recognizesan epitope at 1–313 positions of the N-terminal amino acid sequence ofthe human IL-5R α chain which corresponds to an extracellular regiondefective in the transmembrane region and onwards react specificallywith a human interleukin-5 receptor α chain upon immunocyte staining andinhibit the biological activity of interleukin-5. These antibodies canbe used to diagnose and treat the aforementioned allergic diseases.

Hence, the present invention provides antibodies which reactspecifically with a human IL-5R α chain. The antibodies of the presentinvention include monoclonal antibodies, humanized antibodies, singlechain antibodies, disulfide stabilized antibodies and the like. Theantibodies of the present invention may be of any kind, provided thatthey react specifically with a hIL-5R α chain. Those produced by themethod explained below are preferred. Briefly, hIL-5R α protein isprepared as an antigen and applied to immunize animals such as mice,rats, hamsters, rabbits and the like used to prepare hybridomas, therebyinducing to plasma cells having an antigen specificity. The plasma cellsare fused with mycloma cells to prepare hybridomas which can producemonoclonal antibodies, and the hybridomas are cultured to obtain thedesired anti-IL-5R α monoclonal antibodies. Any monoclonal antibody canbe used so long as it recognizes an epitope at 1–313 positions from theN-terminal amino acid of a human IL-5R α chain and reacts specificallywith the human IL-5R α chain upon immunocyte staining. Alternatively,any monoclonal antibody can be used so long as it recognizes an epitopeat 1–313 positions from the N-terminal amino acid of the human IL-5R αchain and inhibits the biological activity of human IL-5. The formermonoclonal antibodies are exemplified by monoclonal antibody KM1257produced by hybridoma KM1257 (FERM BP-5133). The latter monoclonalantibodies are exemplified by KM1259 produced by hybridoma KM1259 (FERMBP-5134) and KM1486 produced by hybridoma KM1486 (FERM BP-5651). FERMBP-5134 and FERM BP-5651 have been deposited on Jun. 13, 1995 and Sep.3, 1996, respectively, under the terms and conditions of the BudapestTreaty with the International Patent Organism Depository, NationalInstitute of Advanced Industrial Science and Technology.

The monoclonal antibodies of the present invention react immunologicallywith a human IL-5R α chain, a cell having a human IL-5R α chainexpressed on the surface, human eosinophil and the like. The monoclonalantibodies of the present invention react immunologically with a solublehuman IL-5R α chain. Hence, the present invention also provides a methodfor immunologically detecting and determining a human IL-5R α chain, acell having a human IL-5R α chain expressed on the surface, humaneosinophil and a soluble human IL-5R α chain. The results of thedetection and determination can be used in the diagnosis and treatmentof allergic diseases such as chronic bronchial asthma, atopic dermatitisand the like.

The present invention also provides humanized antibodies that havelesser side effects with a prolonged half-life than the monoclonalantibodies and which inhibit the biological activity of IL-5 in a moredesired way as therapeutics. The term “humanized antibody” of thepresent invention is the general term for human chimeric antibodies andhuman CDR-grafted antibodies.

The term “human chimeric antibody” means an antibody consisting of avariable region in a heavy chain (hereinafter referred to as “VH”) and avariable region in a light chain (hereinafter referred to as “VL”) of anon-human animal antibody, as well as a constant region in a heavy chain(hereinafter referred to as “CH”) and a constant region in a light chain(hereinafter referred to as “CL”) of a human antibody. The term “humanCDR-grafted antibody” means an antibody in which CDR sequences of VH andVL of a human antibody are replaced with CDR sequences of VH and VL of anon-human animal antibody, respectively. An anti-hIL-5R α chain humanchimeric antibody which inhibits the biological activity of IL-5 can beexpressed and produced by a process comprising the steps of obtainingcDNAs encoding VH and VL from a hybridoma producing an antibody whichcan inhibit the biological activity of IL-5, inserting the respectivecDNAs into a vector for expression in animal cells which contains a geneencoding human antibody CH and human antibody CL to thereby construct ahuman chimeric antibody expression vector and transfecting theexpression vector into an animal cell. The human chimeric antibody andhuman CDR-grafted antibody of the present invention may be in anyimmunoglobulin (Ig) classes and are preferably in a class of IgG. Inaddition, any C region of IgG subclasses of immunoglobulin such as IgG1,IgG2, IgG3 and IgG4 can be used.

Examples of the human chimeric antibody of the present invention includean antibody of which the VH contains the amino acid sequence of SEQ IDNO: 27, CH is human antibody IgG1, VL contains the amino acid sequenceof SEQ ID NO: 29, and CL is human antibody κ. A specific example is anantibody designated as “KM1399”. A specific example of the humanchimeric antibody of which the CH is human antibody IgG4 is an antibodydesignated as “KM7399”. KM1399 can be produced, for example, bytransformant KM1399 (FERM BP-5650). KM7399 can be produced, for example,by transformant KM7399 (FERM BP-5649).

The anti-hIL-5R α chain human CDR-grafted antibody which inhibits thebiological activity of IL-5 can be expressed and produced by a processcomprising the steps of constructing cDNAs encoding a V region in whichCDR sequences of VH and VL of any human antibody are replaced with CDRsequences of VH and VL, respectively, of a non-human animal antibodywhich can inhibit the biological activity of IL-5, inserting therespective cDNAs into a vector for expression in animal cells whichcontains a gene encoding human antibody CH and human antibody CL tothereby construct a human CDR-grafted antibody expression vector, andtransfecting the expression vector into an animal cell. Examples of thehuman CDR-grafted antibody of the present invention include an antibodyof which the VH contains the amino acid sequence of SEQ ID NO: 83, CH ishuman antibody IgG1, VL contains the amino acid sequence of SEQ ID NO:71, and CL is human antibody κ. A specific example is an antibodydesignated as “KM8399”. A specific example of the human CDR-graftedantibody of which the CH is human antibody IgG4 is an antibodydesignated as “KM9399”. KM8399 can be produced, for example, bytransformant KM8399 (FERM BP-5648). KM9399 can be produced, for example,by transformant KM9399 (FERM BP-5647).

The humanized antibody of the present invention reacts immunologicallywith a human IL-5R α chain, a cell having a human IL-5R α chainexpressed on the surface, human eosinophil and the like. Hence, thehumanized antibody of the present invention can be used in the diagnosisand treatment of allergic diseases such as chronic bronchial asthma,atopic dermatitis and the like.

In addition, the present invention provides single chain antibodies(single chain Fv; hereinafter referred to as “scFv”) and disulfidestabilized antibodies (disulfide stabilized Fv; hereinafter referred toas “dsFv”) which exhibit an ability to bind to a human IL-5R α chain.

The term “single chain antibody (scFv)” means a polypeptide representedby formula VH-L-VL or VL-L-VH in which a single chain of VH and a singlechain of VL are linked by an appropriate peptide linker (hereinafterreferred to as “L”). Any anti-human IL-5R α chain monoclonal antibodiesor human CDR-grafted antibodies can be used as VH and VL in the scFv ofthe present invention.

The term “disulfide stabilized antibody (dsFv)” means an antibodyprepared by binding through a disulfide bond two polypeptides in whicheach one of the amino acid residues in VH and VL is replaced withcysteine residues. The amino acid residues to be replaced with cysteineresidues can be selected on the basis of a presumed steric structure ofan antibody in accordance with the method described by Reiter et al.(Protein Engineering,7, 697 (1994)). Either a mouse anti-human IL-5R αchain monoclonal antibodies or a human CDR-grafted antibodies can beused as VH or VL in the disulfide stabilized antibody of the presentinvention.

The single chain antibody which has an ability to bind to a human IL-5Rα chain can be expressed and produced by a process comprising the stepsof obtaining cDNA encoding VH and VL from a hybridoma which produces anantibody reactive with the human IL-5R α chain, constructing a singlechain antibody expression vector, and transfecting the expression vectorinto an E. coli, yeast or animal cell. Examples of the monoclonalantibody-derived single chain antibody of the present invention includean antibody of which the VH contains the amino acid sequence of SEQ IDNO: 27 and VL contains the amino acid sequence of SEQ ID NO: 29.Examples of the human CDR-grafted antibody-derived single chain antibodyof the present invention include an antibody of which the VH containsthe amino acid sequence of SEQ ID NO: 83 and VL contains the amino acidsequence of SEQ ID NO: 71.

The disulfide stabilized antibody which has an ability to bind to ahuman IL-5R α chain can be expressed and produced by a processcomprising the steps of obtaining cDNA encoding VH and VL from ahybridoma which produces an antibody reactive with the human IL-5R αchain, inserting the cDNA into an appropriate expression vector, andtransfecting the expression vector into an E. coli, yeast or animalcell. Examples of the monoclonal antibody-derived single chain antibodyof the present invention include an antibody of which the VH containsthe amino acid sequence of SEQ ID NO: 27 and VL contains the amino acidsequence of SEQ ID NO: 29. Examples of the human CDR-graftedantibody-derived disulfide stabilized antibody of the present inventioninclude an antibody of which the VH contains the amino acid sequence ofSEQ ID NO: 83 and VL contains the amino acid sequence of SEQ ID NO: 71.

A method for producing an anti-human IL-5R α chain monoclonal antibodywhich reacts specifically with a human IL-5R α chain or which inhibitsthe biological activity of human IL-5, and a method for producing ananti-human IL-5R α chain humanized antibody, an anti-human IL-5R α chainsingle chain antibody and an anti-human IL-5R α chain disulfidestabilized antibody all of which inhibit the biological activity ofhuman IL-5, as well as a method for detecting and determining a humaninterleukin-5 receptor α chain by means of said antibodies will now beexplained in detail.

DETAILED DESCRIPTION OF THE INVENTION

1. Production of Anti-hIL-5R α Monoclonal Antibody

(1) Preparation of Antigen

A cell having hIL-5R α expressed on the cell surface or a cell membranefraction thereof, or an hIL-5R α-expressing cell CTLL-2 (h5 R) or a cellmembrane fraction thereof can be used as an antigen for producing ananti-hIL-5R α monoclonal antibody. CTLL-2 (h5 R) is an hIL-5Rα-expressing cell which was created by inserting a cDNA encoding a fulllength sequence of a pre-cloned hIL-5Rα (J. Exp. Med., 175, 341 (1992))into an expression vector for animal cells such as pCAGGS (Gene,108, 193(1991)) and transfecting the expression vector into murine T cell lineCTLL-2.

For expression in a prokaryotic host cell such as E. coli, a full lengthor partial fragment of cDNA encoding hIL-5R α can be inserted into anexpression vector such as commercially available pGEX (Pharmacia), pETsystem (Novagen), pMKex1 to be described in section (11) of Example 1below or the like and the full length hIL-5R α sequence or a partialfragment thereof can be expressed either as such or as a fusion protein.After disruption of the cell, the protein expressed by E. coli can bepurified by SDS-polyacrylamide electrophoresis, affinity chromatographybased on the nature of the fusion protein, or the like.

In the method of expressing the full length IL-5R α sequence or apartial fragment thereof either as such or as a fusion protein,eukaryotic host cells such as insect cells, mammalian cells and the likecan be used.

In the case of using a mammalian cell, a full length or a partialfragment of cDNA encoding hIL-5R α is inserted into a vector such aspAGE107 (Cytotechnology, 3, 133 (1990)), pAGE103 (J. Biochem.,101, 1307(1987)), pAGE210 to be described in section (1) of Example 1 below orthe like to thereby construct an expression vector for the protein. Inorder to express efficiently the full length hIL-5R α sequence encodedby the cDNA or a partial fragment thereof either as such or as a fusedprotein, the nucleotide sequence encoding a signal peptide in the cDNAis preferably replaced by the nucleotide sequence encoding a signalpeptide of a protein which can be expressed at a high level in aeukaryotic host cell. Known signal peptides of proteins including thoseof human growth hormone, anti-ganglioside GD3 chimeric antibody KM871(Kokai No. 304989/93) and the like are preferably used.

The thus constructed expression vector can be transfected into hostcells by a known method such as electroporation (Kokai No. 257891/90;Cytotechnology, 3, 133 (1990)), lipofectin method (Proc. Natl. Acad.Sci., 84, 7413 (1987)) or the like. The cultivation of the cells in anappropriate medium can result in the production of the full lengthhIL-5R α sequence or a partial fragment thereof either as such or as afusion protein in the cells or the culture supernatant. A serum-freemedium is preferably used because it can facilitate the purification ofthe partial fragment or fusion protein of hIL-5R α produced in theculture supernatant.

In the case of using an insect cell, a full length or a partial fragmentof cDNA encoding hIL-5R α is inserted using a Baculo Gold Starter Kit(Pharmingen) to prepare a recombinant baculovirus and insect cells ofSf9, Sf21 (Pharmingen) or the like are infected with the recombinantvirus such that the full length hIL-5R α sequence or a partial fragmentthereof is produced either as such or as a fusion protein in the cellsor the culture supernatant (Bio/Technology, 6, 47 (1988)).

The full length hIL-5R α sequence or a partial fragment or fusionprotein thereof produced by the animal or insect cells can be purifiedfrom the culture supernatant or the like by a known method of proteinpurification such as salting-out, affinity chromatography, ion-exchangechromatography or the like and can be used as an antigen. Particularlyin the case where the hIL-5R α is produced as a fusion protein with aconstant region of immunoglobulin, it is preferably purified using anaffinity column having fixed thereto protein A which has specificaffinity for the constant region of immunoglobulin.

(2) Immunization of Animal and Preparation of Antibody-Producing Cells

Any animal such as mice, rats, hamsters, rabbits and the like can beused as animals to be immunized, provided that they can be used toprepare hybridomas. An embodiment in which mice or rats are used will beexplained herein. Mice and rats of 3–20 weeks old are immunized withshIL-5R α or CTLL-2 which have hIL-5R α expressed on the surface (J.Exp. Med., 177, 1523 (1993)) as an antigen and antibody-producing cellsare collected from the spleens, lymph nodes and peripheral blood of theanimals. Immunization is performed by administering the animals with theantigen together with an appropriate adjuvant such as complete Freund'sadjuvant or a combination of aluminum hydroxide gel and pertussisvaccine either subcutaneously, intravenously or intraperitoneally. Theantigen is administered 5–10 times at intervals of 1–2 weeks after thefirst administration. Blood is collected from the ophthal venous plexusat day 3–7 after each administration and the serum is examined for areactivity with the antigen by enzyme immunoassay (“Enzyme Immunoassay(ELISA)”, published by Igakushoin, 1976).

A mouse or rat whose serum shows a satisfactory antibody titer toshIL-5R α or the cells which have hIL-5R α expressed on the surface,which are used for immunization, can be used as a source ofantibody-producing cells.

In order to perform fusion of a spleen cell with a mycloma cell, thespleen is removed from the immunized mouse at day 3–7 after the finaladministration of the antigenic substance and spleen cells arecollected. The spleen is sliced in an MEM medium (NissuiPharmaceuticals) and dispersed with a pair of tweezers. Aftercentrifugation (1,200 rpm, 5 min), the supernatant is removed. Theprecipitate is treated with a Tris-ammonium chloride buffer (pH 7.65)for 1–2 minutes to remove erythrocytes and washed with MEM medium 3times to prepare splenocytes for use in cell fusion.

(3) Preparation of Myeloma Cells

An established cell line from a mouse or a rat is used as a myelomacell. Examples include myeloma cell lines P3-X63Ag8-U1 (P3-U1) (Curr.Topics Microbiol. Immunol., 81, 1 (1978); Europ. J. Immunol., 6, 511(1976)), SP2/0-Ag14 (SP-2) (Nature, 276, 269 (1978)), P3-X63-Ag8653(653) (J. Immunol., 123, 1548 (1979)) and P3-X63-Ag8 (X63) (Nature, 256,495 (1975)) which are derived from 8-azaguanine-tolerant mice (BALB/c).These cell lines can be subcultured in 8-azaguanine medium which isRPMI-1640 medium supplemented with glutamine (1.5 mM), 2-mercaptoethanol(5×10⁻⁵ M), gentamicin (10 μg/ml) and fetal calf serum (FCS) (CSL, 10%)(hereinafter referred to as “normal medium”), which is furthersupplemented with 8-azaguanine (15 μg/ml). They should be subcultured ina normal medium 3–4 days before cell fusion to ensure a cell count of atleast 2×10⁷ cells on the day of cell fusion.

(4) Cell Fusion

The antibody-producing cells described in 1 (2) and the myeloma cellsdescribed in 1 (3) are washed thoroughly with MEM medium or PBS (1.83 gof disodium phosphate, 0.21 g of monopotassium phosphate, 7.65 g ofsodium chloride, 1 L of distilled water, pH 7.2). These cells are mixedsuch that a cell count ratio of the antibody-producing cells to themyeloma cells is 5–10:1. After centrifugation (1,200 rpm, 5 min), thesupernatant is removed. The precipitated cells are dispersed and a mixedsolution composed of ethylene glycol-1000 (PEG-1000)(2 g), MEM (2 ml)and dimethyl sulfoxide (DMSO) (0.7 ml) is then added to the cells in anamount of 0.2–1 ml/10⁸ antibody-producing cells while stirring. An MEMmedium (1–2 ml) is added several times at intervals of 1–2 minutes andan additional MEM medium is then added such that the total volume is 50ml. After centrifugation (900 rpm, 5 min), the supernatant is removed.The cells are dispersed gently and then suspended gently in 100 ml of aHAT medium (a normal medium supplemented with 10⁻⁴ M hypoxanthine,1.5×10⁻⁵ M thymidine and 4×10⁻⁷ M aminopterin) by suction and blowoffwith a pipette.

The cell suspension is dispensed in a 96-well culture plate in an amountof 100 μl/well and cultured in a 5% CO₂ incubator at 37° C. for 7–14days.

After the cultivation, an aliquot of the culture supernatant is examinedby enzyme immunoassay to be described in 1 (5) to select a well that isreactive specifically with a recombinant protein such as a fusionprotein with shIL-5R α or hIL-5R α described in 1 (1). Subsequently,cloning by limiting dilution is repeated twice. An aminopterin-free HATmedium is used in the first cloning and a normal medium in the secondcloning. A cell exhibiting a high antibody titer stably is selected as ahybridoma cell line which produces a mouse or rat anti-hIL-5R αmonoclonal antibody.

(5) Selection of Mouse or Rat Anti-Human IL-5R α Monoclonal Antibody

A mouse or rat anti-hIL-5R α monoclonal antibody-producing hybridoma isselected in accordance with a method as described in Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory, Chapter 14 (1988) bythe measurement method described below. By the method, the activity ofan anti-hIL-5R α antibody in the culture supernatant of thetransformants producing an anti-hIL-5R α humanized antibody, a singlechain antibody or a disulfide stabilized antibody which are to bedescribed below or the activities of all purified anti-hIL-5R αantibodies can be determined.

An appropriate plate is coated with shIL-5R α or a recombinant proteinsuch as a fusion protein with hIL-5R α described in 1 (1). The plate isreacted with a primary antibody which is the hybridoma culturesupernatant or the purified antibody to be obtained in 1 (6) and reactedwith a secondary antibody which is an anti-mouse immunoglobulin antibodyor an anti-rat immunoglobulin antibody which is labeled with biotin, anenzyme, a chemiluminescent substance or a radioactive compound.Subsequently, a reaction is performed in accordance with the specifickind of the label, whereby a hybridoma that is reactive specificallywith hIL-5R α is selected as a hybridoma producing a mouse anti-hIL-5R αmonoclonal antibody.

If the culture supernatant of the transformants producing an anti-hIL-5Rα humanized antibody, a single chain antibody or a disulfide stabilizedantibody, or an antibody purified therefrom is reacted as a primaryantibody, an anti-human immunoglobulin antibody labeled with biotin, anenzyme, a chemiluminescent substance or a radioactive compound is usedas a secondary antibody and a reaction is performed in accordance withthe specific kind of the label for detection.

An appropriate plate is coated with shIL-5R α or a recombinant proteinsuch as a fusion protein with a recombinant protein hIL-5R α describedin 1 (1). Any one of the hybridoma culture supernatant, the culturesupernatant of the transformants producing an anti-hIL-5R α humanizedantibody, a single chain antibody or a disulfide stabilized antibody, oran antibody purified therefrom is mixed and reacted with human IL-5labeled with biotin, an enzyme, a chemiluminescent substance or aradioactive compound. Subsequently, a reaction is performed inaccordance with the specific kind of the label so as to determine anactivity in inhibiting the binding of human IL-5 to human IL-5R α. Thismethod is used to screen hybridomas for selection of one having a highinhibitory activity against human IL-5.

(6) Production of Mouse or Rat Monoclonal Antibody

A 8–10 week-old mouse or nude mouse is treated with pristane. Morespecifically, the mouse is administered intraperitoneally with pristane(2,6,10,14-tetramethylpentadecane, 0.5 ml) and bred for 2 weeks. Themouse is administered intraperitoneally with the mouse or ratanti-hIL-5R α monoclonal antibody-producing hybridoma cell lines (asobtained in 1 (3)) in an amount of 2×10⁷–5×10⁶ cells/mouse. Thehybridoma caused ascites tumor after 10–21 days administration. Theascites is collected from the mouse and centrifuged (3,000 rpm, 5 min)to remove a solid portion. The precipitate is salted out and applied toa column for a caprylic acid precipitation, or a DEAE-Sepharose column,a protein A-column or a Cellulofine GSL2000 column (BiochemicalIndustry) to collect IgG or IgM fractions. These fractions are used as apurified monoclonal antibody.

The subclass of the antibody is determined using a mouse or ratmonoclonal antibody typing kit. The mass of the protein is calculated bya Lowry method or from the absorbance at 280 nm.

2. Production of Anti-Human IL-5R α Humanized Antibody

(1) Construction of Humanized Antibody Expression Vector

In order to produce a humanized antibody from a non-human animalantibody, a humanized antibody expression vector is prepared. Thehumanized antibody expression vector is a vector for expression inanimal cells into which a gene encoding CH and CL, C regions of a humanantibody, have been transfected. Such an expression vector isconstructed by inserting two genes, one encoding CH of a human antibodyand the other encoding CL of a human antibody, into an expression vectorfor animal cells. Any C regions of a human antibody such as Cγ1 and Cγ4of a human antibody H chain, Cκ of a human antibody L chain and the likecan be used. A chromosomal DNA consisting of an exon(s) and an intron(s)or cDNA can be used as a gene encoding a C region of a human antibody.Any expression vectors can be used as expression vectors for animalcells, provided that they can incorporate and express a gene encoding aC region of a human antibody. Examples are pAGE107 (Cytotechnology, 3,133 (1990)), pAGE103 (J. Biochem., 101, 1307 (1987)), pHSG274 (Gene, 27,223 (1984)), pKCR (Proc. Natl. Acad. Sci., 78, 1527 (1981)) and pSG1βd2-4 (Cytotechnology, 4. 173 (1990)). A promoter and an enhancer to beused in preparation of an expression vector for animal cells areexemplified by an SV40 early promoter and enhancer (J. Biochem.,101,1307 (1987)), a Moloney mouse leukemia virus LTR promoter and enhancer(Biochem. Biophys. Res. Commun., 149, 960 (1987)), an immunoglobulin Hchain promoter (Cell, 41, 479 (1985)) and enhancer (Cell, 33, 717(1983)), and the like.

The humanized antibody expression vector may be either of a type inwhich a gene encoding an antibody H chain and a gene encoding anantibody L chain exist on separate vectors or of a type in which bothgenes exist on the same vector (tandem type). In terms of ease ofconstruction of a humanized antibody expression vector, easiness ofintroduction into animal cells, balance between the expression amountsof antibody H and L chains in the animal cells and for other reasons, atandem type of humanized antibody expression vector is more preferred(J. Immunol. Methods, 167, 271 (1994)).

(2) Preparation of cDNA Encoding VH and VL of Non-Human Animal Antibody

cDNA encoding VH and VL of a non-human animal antibody such as a mouseanti-human IL-5R α chain monoclonal antibody is obtained, for example,as follows:

mRNA is extracted from an anti-human IL-5R α chain monoclonalantibody-producing cell such as a mouse anti-human IL-5R α chainantibody-producing hybridoma and used to synthesize cDNA. Thesynthesized cDNA is inserted into a vector such as a phage or a plasmidto prepare a cDNA library. From the library, with a portion in a V or Cregion of a non-human animal antibody such as a mouse antibody beingused as a probe, a recombinant phage or plasmid which contains cDNAencoding VH and a recombinant phage or plasmid which contains cDNAencoding VL are isolated separately. The full nucleotide sequences of VHand VL of an antibody of interest which exist on the recombinant phageor plasmid are determined and the full amino acid sequences of the VHand VL are deduced from the nucleotide sequences.

(3) Construction of Human Chimeric Antibody Expression Vector

A human chimeric antibody expression vector can be constructed byinserting cDNA encoding VH and VL of a non-human animal antibody in aregion upstream of the gene encoding CH and CL of the human antibody onthe humanized antibody expression vector which has been constructed in 2(1). For example, a restriction enzyme recognition site for cloning ofcDNA encoding VH and VL of a non-human animal antibody is createdpreliminarily in a region upstream of a gene encoding CH and CL of thehuman antibody on a chimeric antibody expression vector. At the cloningsite, cDNA encoding a V region of a non-human animal antibody isinserted through a synthetic DNA (see below) to prepare a human chimericantibody expression vector. The synthetic DNA consists of a nucleotidesequence at the 3′ end of a V region of the non-human animal and anucleotide sequence at the 5′ end of a C region of the human antibodyand are prepared by a DNA synthesizer such that it has appropriaterestriction enzyme sites at both ends.

(4) Identification of CDR Sequences of Non-Human Animal Antibody

VH and VL which form an antigen-binding site of an antibody consist of 3complementarity determining regions (CDRs) having a wide variety ofsequences which link the VH and VL to 4 framework regions (hereinafterreferred to as R regions”) having relatively conserved sequences(Sequences of Proteins of Immunological Interest, US Dept. Health andHuman Services, 1991). The amino acid sequence of the respective CDR(CDR sequence) can be identified by comparison with the amino acidsequences of V regions of known antibodies (Sequences of Proteins ofImmunological Interest, US Dept. Health and Human Services, 1991).

(5) Construction of cDNA Encoding V Region of Human CDR-Grafted Antibody

cDNA encoding VH and VL of a human CDR-grafted antibody can be obtainedas follows:

In the first step, for each of VH and VL, the amino acid sequence of FRin a V region of a human antibody to which CDR in a V region of anon-human animal antibody of interest is to be grafted is selected. Anyamino acid sequences of FRs in V regions derived from human antibodiescan be used as the amino acid sequences of FRs in V regions of humanantibodies. For example, the amino acid sequences of FRs in V regions ofhuman antibodies recorded in Protein Data Bank and amino acid sequencescommon to subgroups of FRs in V regions of human antibodies (Sequencesof Proteins of Immunological Interest, US Dept. Health and HumanServices, 1991) can be used. In order to produce a human CDR-graftedantibody having an excellent activity, an amino acid sequence havinghigh homology with the amino acid sequence of a V region of a non-humananimal antibody of interest is desired. In the second step, a DNAsequence encoding the selected amino acid sequence of FR in a V regionof a human antibody is ligated to a DNA sequence encoding the amino acidsequence of CDR in a V region of a non-human animal antibody and a DNAsequence encoding the amino acid sequences of VH and VL is designed. Inorder to obtain a DNA sequence designed to construct a CDR-graftedantibody variable region gene, several synthetic DNAs are designed foreach strand such that the full DNA sequence is covered. Using thesynthetic DNAs, polymerase chain reaction (hereinafter referred to asCR”) is performed. For each strand, preferably 6 synthetic DNAs aredesigned in view of the reaction efficiency of PCR and the lengths ofDNAs which can be synthesized. After the reaction, amplified fragmentsare subcloned into appropriate vectors and their nucleotide sequencesare determined, thereby obtaining a plasmid which contains cDNA encodingthe amino acid sequence of a V region of each strand of a humanCDR-grafted antibody of interest. Alternatively, cDNA encoding the aminoacid sequence of a V region of each strand of a human CDR-graftedantibody of interest may be constructed by synthesizing the fullsequences of sense and antisense strands using synthetic DNAs consistingof about 100 bases and subjecting them to annealing and ligation.

(6) Modification of the Amino Acid Sequence of V Region of HumanCDR-Grafted Antibody

It is known that if a human CDR-grafted antibody is prepared by simplygrafting only CDR in a V region of a non-human animal antibody ofinterest between FRs in a V region of a human antibody, its activity islower than that of the original non-human animal antibody(BIO/TECHNOLOGY, 9, 266 (1991)). Hence, among the amino acid sequencesof FR in a V region of a human antibody, an amino acid residue whichtakes part in direct binding to an antigen, an amino acid residue whichinteracts with an amino acid residue in CDR, or an amino acid residuewhich may take part in the maintenance of the steric structure of anantibody is modified to an amino acid residue that is found in theoriginal non-human animal antibody such that the activity of the humanCDR-grafted antibody is increased. For efficient identification of theamino acid residue, the steric structure of an antibody is constructedand analyzed by X-ray crystallography, computer-modeling or the like.However, no method for producing a human CDR-grafted antibody which canbe applied to any antibodies has yet been established and, therefore,various attempts must currently be made on a case-by-case basis.

The modification of the selected amino acid sequence of FR in a V regionof a human antibody can be accomplished using various primers formutation by PCR described in 2 (5). Amplified fragments obtained by thePCR are subcloned into appropriate vectors and their nucleotidesequences are determined, thereby obtaining a vector containing cDNAinto which a mutation of interest has been introduced (hereinafterreferred to as “amino acid sequence-replaced vector”).

Alternatively, the modification of an amino acid sequence in a narrowregion may be accomplished by a PCR-mutagenesis method using primers formutation consisting of 20–35 bases. More specifically, a sense mutationprimer and an antisense mutation primer which consist of 20–35 bases andwhich contain DNA sequences encoding the amino acid residue to bemodified are synthesized and used to perform 2-step PCR using as atemplate a plasmid which contains cDNA encoding the amino acid sequenceof a V region which is to be modified. The finally amplified fragmentsare subcloned into appropriate vectors and their nucleotide sequencesare determined, thereby obtaining an amino acid sequence-modified vectorcontaining cDNA into which a mutation of interest has been introduced.

(7) Construction of Human CDR-Grafted Antibody Expression Vector

A human CDR-grafted antibody expression vector can be constructed byinserting the cDNA encoding VH and VL of the human CDR-grafted antibodyobtained in 2 (5) and 2 (6) in a region upstream of the gene encoding CHand CL of the human antibody in the humanized antibody expression vectordescribed in 2 (1). For example, if recognition sites for appropriateenzymes are introduced at the ends of the 5′ and 3′ terminal syntheticDNAs during PCR for construction of cDNA encoding the amino acidsequences of VH and VL of the human CDR-grafted antibody, the cDNA canbe inserted in a region upstream of a gene encoding a C region of adesired human antibody such that it is expressed in an appropriate form.

(8) Transient Expression of Humanized Antibodies and Evaluation of TheirActivities

In order to evaluate the activities of a wide variety of humanizedantibodies efficiently, the human chimeric antibody expression vectordescribed in 2 (3), and the human CDR-grafted antibody expression vectordescribed in 2 (7) or their modified vectors may be transfected intoCOS-7 cells (ATCC CRL1651) and humanized antibodies expressedtransiently (Methods in Nucleic Acids Res., CRC Press, p.283, 1991),followed by determination of their activities.

Examples of the method for transfecting the expression vector into aCOS-7 cell include a DEAE-dextran method (Methods in Nucleic Acids Res.,CRC Press, p.283, 1991), a lipofection method (Proc. Natl. Acad. Sci.,84, 7413 (1987)) and the like.

After transfection of the vector, the activities of the humanizedantibodies in the culture supernatant can be determined by the enzymeimmunoassay (ELISA) described in 1 (5) and the like.

(9) Stable Expression of Humanized Antibodies and Evaluation of TheirActivities

Transformants which produce a humanized antibody stably can be obtainedby transfecting into appropriate host cells the human chimeric antibodyexpression vector described in 2 (3) and the human CDR-grafted antibodyexpression vector described in 2 (7).

Examples of the method for transfecting the expression vector into hostcells include electroporation (Kokai No. 257891/90, Cytotechnology, 3,133 (1990)) and the like.

Any cells can be used as host cells into which the humanized antibodyexpression vector is to be transfected, provided that they can express ahumanized antibody. Examples are mouse SP2/0-Ag14 cell (ATCC CRL1581),mouse P3X63-Ag8.653 cell (ATCC CRL1580), CHO cells which are detectivein dihydrofolate reductase gene (hereinafter referred to as “DHFR gene”)(Proc. Natl. Acad. Sci.,77, 4216 (1980)) and rat YB2/3HL.P2.G11.16Ag.20cell (ATCC CRL1662, hereinafter referred to as “YB2/0 cell”).

After transfection of the vector, transformants which express ahumanized antibody stably are selected in accordance with the methoddisclosed in Kokai No. 257891/90, using an RPMI1640 medium containingG418 and FCS. The humanized antibody can be produced and accumulated ina culture medium by culturing the selected transformants in a medium.The activity of the humanized antibody in the culture medium isdetermined by the method described in 1 (5) or the like. The productionof the humanized antibody by the transformants can be increased by themethod described in Kokai No. 257891/90, utilizing a DHFRgene-amplification system or the like.

The humanized antibody can be purified from the culture supernatant ofthe transformants by using a protein A column (Antibodies, A LaboratoryManual, Cold Spring Harbor, Chapter 8, 1988). Any other conventionalmethods for protein purification can be used. For example, the humanizedantibody can be purified by a combination of gel filtration,ion-exchange chromatography, ultrafiltration and the like. The molecularweight of the H chain or L chain of the purified humanized antibody orthe antibody molecule as a whole is determined by polyacrylamide gelelectrophoresis (SDS-PAGE) (Nature, 227, 680, (1970)), western blotting(Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter12, 1988) and the like.

The reactivity of the purified humanized antibody and the inhibitionactivity of the humanized antibody against IL-5 can be determined by themethod described in 1 (5).

(10) Method of Use of Humanized Antibody

The humanized antibody of the present invention can bind specifically toa human IL-5R α chain, thereby inhibiting the biological activity ofIL-5. Hence, the humanized antibody of the present invention is expectedto inhibit the function of eosinophils which are controlled indifferentiation and growth by IL-5. Accordingly, the humanized antibodyof the present invention will be useful in the treatment of diseaseswhere eosinophils are associated with their pathogenesis. Since almostall portions of the humanized antibody of the present invention arederived from the amino acid sequence of a human antibody, it is expectednot only to exhibit immunogenicity in the human body but also tomaintain its effect for a long period of time. The humanized antibody ofthe present invention can be used either alone or in combination with atleast one pharmaceutically acceptable adjuvant. For example, thehumanized antibody is dissolved in physiological saline or an aqueoussolution of glucose, lactose, mannitol or the like to prepare apharmaceutical composition. Alternatively, the humanized antibody islyophilized by a conventional method and sodium chloride is added toprepare an injection in a powder form. If necessary, the presentpharmaceutical composition may contain any additive that is well knownin the field of pharmaceutical preparations such as a pharmaceuticallyacceptable salt and the like.

The present pharmaceutical composition can be administered to mammalsincluding human at a dose of 0.1–20 mg/kg/day of the humanized antibody,which may vary depending on the age and conditions of the patient andthe like. The administration is given once a day (single dose orcontinuous administration), 1–3 times a week or once every 2–3 weeks byintravenous injection.

3. Production of Anti-Human IL-5R α Single Chain Antibody

(1) Construction of Single Chain Antibody Expression Vector

A vector for expression of a single chain antibody of a non-human animalantibody or a single chain antibody of a human CDR-grafted antibody canbe constructed by inserting into a single chain antibody expressionvector the cDNAs encoding VH and VL of a non-human animal antibody or ahuman CDR-grafted antibody which are described in 2 (2), 2 (5) and 2(6). Any expression vectors can be used as single chain antibodyexpression vectors, provided that they can incorporate and express thecDNAs encoding VH and VL of a non-human animal antibody or a humanCDR-grafted antibody. Examples are pAGE107 (Cytotechnology, 3, 133(1990)), pAGE103 (J. Biochem., 101, 1307 (1987)), pHSG274 (Gene, 27, 223(1984)), pKCR (Proc. Natl. Acad. Sci., 78, 1527 (1981)) and pSG1 βd2-4(Cytotechnology, 4. 173 (1990)). A host for use in expressing a singlechain antibody can be selected from among E. coli, yeast and animalcells and the like. In this case, an expression vector which iscompatible with the specific host should be selected. The single chainantibody can be secreted out of the cell and transported into theperiplasm region or retained within the cell by inserting a cDNAencoding an appropriate signal peptide into the expression vector.

A single chain antibody expression vector into which the cDNA encoding asingle chain antibody of interest has been inserted can be constructedby inserting the cDNA encoding a single chain antibody consisting ofVH-L-VL or VL-L-VH (where L is a peptide linker) into the selectedexpression vector in a region downstream of an appropriate promoter anda signal peptide.

The cDNA encoding a single chain antibody can be obtained by linking aVH encoding cDNA to a VL encoding cDNA through a synthetic DNA encodinga peptide linker having recognition sites for appropriate restrictionenzymes at both the ends. It is important to optimize the linker peptidesuch that its addition does not interfere with the binding of VH and VLto an antigen. For example, the linker described by Pantoliano et al.(Biochemistry, 30, 10117 (1991)) and its modified versions may be used.

(2) Expression of Single Chain Antibody and Evaluation of its Activity

A transformant which produces a single chain antibody of interest can beobtained by transfecting the single chain antibody expression vectorconstructed in 3 (1) into an appropriate host cell by electroporation(Kokai No. 257891/90; Cytotechnology, 3, 133 (1990)) or the like. Aftertransfection of the expression vector, the activity of the single chainantibody in the culture supernatant can be determined by the methoddescribed in 1 (5) or the like.

The collection and purification of the single chain antibody of thepresent invention can be accomplished by a combination of knowntechniques. For example, if the single chain antibody is secreted in amedium, it can be concentrated by ultrafiltration and its collection andpurification can be then performed by antigen affinity chromatography orion-exchange chromatography or gel filtration. If the single chainantibody is transported into the periplasm region of the host cell, itcan be concentrated by ultrafiltration following the application of anosmotic shock and its collection and purification can be then performedby antigen affinity chromatography or ion-exchange chromatography or gelfiltration. If the single chain antibody is insoluble and exists as agranule (i.e., inclusion body), its collection and purification can beperformed by lysis of the cell, repeated centrifugation and washing forisolation of the granule, solubilization with guanidine-HCl, anoperation for returning the structure of the single chain antibody to anactive structure and the subsequent purification of an active molecule.

The activity of the purified single chain antibody can be determined bythe method described in 1 (5) or the like.

(3) Method of Using Single Chain Antibody

The single chain antibody of the present invention can bind specificallyto a human IL-5R α chain, and inhibit the biological activity of IL-5.Hence, the single chain antibody of the present invention is expected toinhibit the function of eosinophils which are controlled indifferentiation and growth by IL-5. Accordingly, the single chainantibody of the present invention will be useful in the treatment ofdiseases in which eosinophils are associated with the pathogenesis. Thesingle chain antibody of the present invention can be used either aloneor in combination with at least one pharmaceutically acceptableadjuvant. For example, the single chain antibody is dissolved inphysiological saline or an aqueous solution of glucose, lactose,mannitol or the like to prepare a pharmaceutical composition.Alternatively, the single chain antibody is lyophilized by aconventional method and sodium chloride is added to prepare an injectionin a powder form. If necessary, the present pharmaceutical compositionmay contain any additive that is well known in the field ofpharmaceutical preparations such as a pharmaceutically acceptable saltand the like.

The present pharmaceutical composition can be administered to mammals,including humans, at a dose of 0.1–20 mg/kg/day of the signal chainantibody, which may vary depending on the age and condition of thepatient and the like. The administration is given once a day (singledose or continuous administration), 1–3 times a week or once every 2–3weeks by intravenous injection.

4. Production of Anti-Human IL-5R α Disulfide Stabilized Antibody

(1) Production of Disulfide Stabilized Antibody

A disulfide stabilized antibody can be produced by a process comprisingthe steps of providing cDNAs encoding VH and VL of a non-human animalantibody or cDNAs encoding VH and VL of a human CDR-grafted antibody,modifying the DNA sequence which corresponds to a one-amino acid residueat an appropriate position in the respective cDNA with a DNA sequencecorresponding to a cysteine residue, expressing the modified cDNAs andpurifying the resultant peptide and then forming a disulfide bond. Themodification of an amino acid residue to a cysteine residue can beperformed by a mutagenesis method using PCR described in 2 (5).

A disulfide stabilized antibody H chain expression vector and adisulfide stabilized antibody L chain expression vector can beconstructed by inserting the resulting cDNAs encoding the modified VHand modified VL into appropriate expression vectors. Any expressionvectors can be used as disulfide stabilized antibody expression vectors,provided that they can incorporate and express cDNAs encoding a modifiedVH and a modified VL. For example, pAGE107 (Cytotechnology, 3, 133(1990)), pAGE103 (J. Biochem., 101, 1307 (1987)), pHSG274 (Gene, 27, 223(1984)), pKCR (Proc. Natl. Acad. Sci., 78, 1527 (1981)), pSG1 βd2-4(Cytotechnology, 4. 173 (1990)) and the like can be used. A host used toexpress a disulfide stabilized antibody L chain expression vector and adisulfide stabilized antibody H chain expression vector for formation ofa disulfide stabilized antibody can be selected from among E. coli,yeast and animal cells, and the like. In this case, an expression vectorwhich is compatible with the specific host should be selected. Thedisulfide stabilized antibody can be secreted out of the cell andtransported into the periplasm region or retained within the cell byinserting a cDNA encoding an appropriate signal peptide into theexpression vector.

(2) Expression of Disulfide Stabilized Antibody and Evaluation of itsActivity

A transformant which produces a disulfide stabilized antibody H chain ora disulfide stabilized antibody L chain of interest can be obtained bytransfecting into a host cell the disulfide stabilized antibody H chainexpression vector or the disulfide stabilized antibody L chainexpression vector that were constructed in 4 (1) by electroporation(Kokai No. 257891/90; Cytotechnology, 3, 133 (1990)) or the like. Afterintroduction of the expression vector, the expression of the disulfidestabilized antibody H chain or disulfide stabilized antibody L chain inthe culture supernatant or the like can be confirmed by the methoddescribed in 1 (5).

The collection and purification of the disulfide stabilized antibody Hchain or disulfide stabilized antibody L chain can be accomplished bycombinations of known techniques. For example, if the disulfidestabilized antibody H chain or disulfide stabilized antibody L chain issecreted in a medium, they can be concentrated by ultrafiltration andtheir collection and purification can be then performed by various typesof chromatography or gel filtration. If the disulfide stabilizedantibody H chain or disulfide stabilized antibody L chain is transportedinto the periplasm region of the host cell, they can be concentrated byultrafiltration after the application of an osmotic shock to the celland their collection and purification can be then performed by varioustypes of chromatography or gel filtration. If the disulfide stabilizedantibody H chain or disulfide stabilized antibody L chain is insolubleand exists as a granule (i.e., inclusion body), their collection andpurification can be performed by lysis of the cells, repeatedcentrifugation and washing for isolation of the granule, solubilizationwith guanidine-HCl and subsequent performance of various types ofchromatography or gel filtration.

The purified disulfide stabilized antibody H chain and disulfidestabilized antibody L chain are mixed and subjected to a refoldingprocedure for deriving an active structure (Molecular Immunology, 32,249 (1995)), thereby forming a disulfide bond. Subsequently, the activedisulfide stabilized antibody can be purified by antigen affinitychromatography or ion-exchange chromatography or gel filtration. Theactivity of the disulfide stabilized antibody can be determined by themethod described in 1 (5) or the like.

(3) Method of Use of Disulfide Stabilized Antibody

The disulfide stabilized antibody of the present invention can bindspecifically to a human IL-5R α chain, thereby inhibiting the biologicalactivity of IL-5. Hence, the disulfide stabilized antibody of thepresent invention is expected to inhibit the function of eosinophilswhich are controlled in differentiation and growth by IL-5. Accordingly,the disulfide stabilized antibody of the present invention will beuseful in the treatment of diseases in which eosinophils are associatedwith the pathogenesis. The disulfide stabilized antibody of the presentinvention can be used either alone or in combination with at least onepharmaceutically acceptable adjuvant. For example, the single chainantibody or disulfide stabilized antibody is dissolved in physiologicalsaline or an aqueous solution of glucose, lactose, mannitol or the liketo prepare a pharmaceutical composition. Alternatively, the disulfidestabilized antibody is lyophilized by a conventional method and sodiumchloride is added to prepare an injection in a powder form. Ifnecessary, the present pharmaceutical composition may contain anyadditive that is well known in the field of pharmaceutical preparationssuch as a pharmaceutically acceptable salt and the like.

The present pharmaceutical composition can be administered to mammals,including humans, at a dose of 0.1–20 mg/kg/day of the disulfidestabilized antibody, which may vary depending on the age and conditionof the patient and the like. The administration is given once a day(single dose or continuous administration), 1–3 times a week or onceevery 2–3 weeks by intravenous injection.

5. Method for Detection and Determination of Human Interleukin-5Receptor α Chain Using Anti-Human IL-5R α Antibody

(1) Immunocyte Staining Using Anti-Human IL-5R α Antibody

When immunocytes are suspended cells, they are used as such in thefollowing treatment. When immunocytes are adherent cells, they aredetached with trypsin in EDTA and then used in the following treatment.The immunocytes are suspended in an immunocyte stain buffer (PBScontaining 1% BAS, 0.02% EDTA and 0.05% sodium azide) or the like anddispensed in an amount of 1×10⁵–2×10⁶ cells. The culture supernatant ofthe anti-human IL-5R α monoclonal antibody-producing hybridoma obtainedin 1 (4), the culture supernatant of the anti-human IL-5R α humanizedantibody transformant obtained in 2 (9) or the purified antibodyobtained in 1 (6) or 2 (9), or the product obtained by labeling thepurified antibody with an appropriate labeling substance (e.g., biotin)by a known method (KOUSOKOUTAIHOU (Methods for Enzymes and Antibodies),published by Gakusai Kikaku, 1985) and diluting the labeled antibodywith an immunocyte stain buffer or a 10% animal serum-containingimmunocyte stain buffer to a concentration of 0.1–50 μg/ml is dispensedin an amount of 20–500 μl and reacted on ice for 30 minutes. When theculture supernatant of the mouse anti-human IL-5R α monoclonalantibody-producing hybridoma obtained in 1 (4), the anti-human IL-5R αhumanized antibody transformant obtained in 2 (9) or the purifiedantibody obtained in 1 (6) or 2 (9) has been reacted, the cells arewashed with an immunocyte stain buffer after completion of the reactionand an immunocyte stain buffer containing about 0.1–50 μg/ml of ananti-mouse immunoglobulin antibody, anti-rat immunoglobulin antibody oranti-human immunoglobulin antibody which have been labeled with afluorochrome such as FITC or phycoerythrin is dispensed in an amount of50–500 μl, followed by reaction on ice for 30 minutes in the dark. Whenthe biotin-labeled monoclonal antibody has been reacted, streptoavidinlabeled with a fluorochrome such as FITC or phycoerythrin is dispensedin an amount of 50–500 μl and reaction is performed on ice for 30minutes in the dark. When the monoclonal antibody labeled with afluorochrome such as FITC or phycoerythrin has been reacted, animmunocyte stain buffer containing about 0.1–50 μg/ml of the monoclonalantibody is dispensed in an amount of 50–500 μl and reaction isperformed on ice for 30 minutes in the dark. In each of these cases, thereaction mixture is washed thoroughly with an immunocyte stain bufferafter the reaction and subjected to an analysis with a cell sorter.

(2) Test for Inhibition of Growth of Human IL-5-Dependent Cells UsingAnti-Human IL-5R α Antibody

In order to show the biological inhibition activity of the obtainedanti-human IL-5R α antibody, the effect on the growth of humanIL-5-dependent cells is examined using human IL-5 dependent cells.Examples of the evaluation method include incorporation oftritium-labeled thymidine into cells, color development methods usingcell counting kits and the like. A color development method used in thepresent invention will now be explained.

CTLL-2 (h5R) cells (1×10⁴) are suspended in a normal medium (50 μl) anddispensed in a 96-well culture plate. To the plate are added 25 μl of asolution of the purified antibody (0.01–50 μg/ml) obtained in 1 (6) or 2(9) and a normal medium containing 0.4–40 ng/ml of human IL-5 and themixture is cultured in a 5% CO₂ incubator at 37° C. for 24–72 hours.Subsequently, a cell counting kit solution is added at 10 μl/well andthe cultivation is continued in a 5% CO₂ incubator at 37° C. for 4hours. After completion of the cultivation, the absorbance at 450 nm isdetermined with a microwell plate reader Emax (Molecular Device) and theCTLL-2 (h5R) cell growth-inhibiting activity of the respective antibodyis calculated.

(3) Suppression of Survival of Human Eosinophils by Anti-Human IL-5R αAntibody

Human polymorphonuclear leukocyte fractions which contain eosinophilsare prepared from human peripheral blood with a commercially availablecorpuscle separation medium such as a polymorphprep (Nikomed) or apercoll (Pharmacia). The fractions are suspended in a normal medium andthe resulting cells are dispensed in a 96, 48 or 24-well culture platein an amount of 1×10⁶–1×10⁷ cells/well, followed by addition of humanIL-5 to a final concentration of 0.001–10 ng/ml. The culture supernatantof the anti-human IL-5R α monoclonal antibody-producing hybridomaobtained in 1 (4) or the culture supernatant of the anti-human IL-5R αhumanized antibody transformant obtained in 2 (9) or the purifiedantibody obtained in 1 (6) or 2 (9) is added and the mixture is culturedin a 5% CO₂ incubator at 37° C. for 2–5 days. After completion of thecultivation, a cell sample is prepared from each well and stained byMay-Grunwald-Giemsa staining method (SENSHOKUHOU NO SUBETE (Techniquesfor Staining, published by Ishiyaku Shuppan Cor., Ltd., 1988) or thelike and the percentage of eosinophils is determined. The absence orpresence of the activity of the monoclonal antibody in suppressing theviability enhancement of IL-5-dependent human eosinophils is confirmedby comparing the percentage of eosinophils in the absence of theanti-human IL-5R α antibody with that in the presence of the anti-humanIL-5R α antibody.

(4) Determination of shIL-5R α Using Monoclonal Antibody

A plate is coated with 0.1–50 μg/ml of the purified antibody obtained in1 (6) or 2 (9) as a primary antibody. The coated plate is reacted with0.1–10,000 ng/ml of the purified shIL-5R α obtained in 1 (1) or a samplesuch as human serum. The plate is washed thoroughly and then reactedwith a secondary antibody which is an anti-human IL-5R α antibodyrecognizing an epitope other than that recognized by the anti-humanIL-5R α antibody which was selected for use as the primary antibody fromthe purified antibodies obtained in 1 (6) or 2 (9). The secondaryantibody was labeled with biotin, an enzyme, a chemiluminescentsubstance, a radioactive compound or the like prior to the reaction.Subsequently, a reaction is performed in accordance with the label. Acalibration curve is constructed on the basis of the reactivity with thepurified shIL-5R and the concentration of shIL-5R in the sample iscalculated.

(5) Detection of shIL-5R α by Western Blotting

The purified shIL-5R α obtained in 1 (1) is subjected to SDSpolyacrylamide electrophoresis (SDS-PAGE) and then blotted on apolyvinylidene difluoride membrane (hereinafter referred to as “PVDFmembrane”, Millipore). The PVDF membrane is immersed in PBS supplementedwith 1–10% bovine serum albumin (BSA) and left to stand at 4° C.overnight for blocking, followed by thorough washing with PBS containing0.05% Tween. The PVDF membrane is immersed in the culture supernatant ofthe hybridoma obtained in 1 (5) or a solution of the purified antibodyobtained in 1 (6) at room temperature for 2 hours and washed thoroughlywith PBS containing 0.05% Tween. The PVDF membrane is immersed in asolution of an anti-mouse immunoglobulin antibody or anti-ratimmunoglobulin antibody as a secondary antibody at room temperature for1 hour and washed thoroughly with PBS containing 0.05% Tween. Thesecondary antibody was labeled preliminarily with biotin, an enzyme, achemiluminescent substance, a radioactive compound or the like. Afterremoving the washing solution completely, a reaction is performed inaccordance with the label on the secondary antibody and a check is madefor the reactivity with a protein which agrees in the molecular weightto the purified shIL-5R α.

(6) Immunoprecipitation of shIL-5R α

An anti-mouse immunoglobulin antibody or anti-rat immunoglobulinantibody is diluted 10–1000 fold with PBS or other buffer. The dilutionsare dispensed in a 96-well ELISA plastic plate at 50–200 μl/well andleft to stand at 4° C. overnight or at room temperature for at least 2hours, whereby they are adsorbed on the plate. The plate is washed withPBS. PBS containing 1–10% BSA and the like is dispensed in the plate at300 μl/well and left to stand at 4° C. overnight or at room temperaturefor at least 30 minutes to achieve blocking. The plate is washed withPBS. The culture supernatant of the hybridoma obtained in 1 (5) or asolution of the purified antibody obtained in 1 (6) (0.01–50 μg/ml) isadded at 50–200 μl/well and left to stand at 4° C. overnight, therebyadsorbing the antibody on the plate. After the plate is washed, theshIL-5R α obtained in 1 (1) is diluted with PBS or the like containing1% BSA to a concentration of 0.1–100 μg/ml and the dilutions aredispensed at 50–200 μl/well, followed by reaction at 4° C. overnight.After the plate is washed with PBS or the like containing 0.05% Tween, a1×–5×sample buffer for SDS-PAGE is dispensed at 50–200 μl/well andshaken at room temperature for at least 30 minutes. After optionaldilution with PBS, the solution is added to each lane in an amount of5–25 μl and subjected to SDS-PAGE, followed by blotting on a PVDFmembrane or the like by a conventional method. The PVDF membrane issubjected to western blotting as described in 5 (5), thereby detectingshIL-5R α.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows steps for constructing plasmid pAGE210.

FIG. 2 shows the restriction map of plasmid pCAGGS-h5R.25.

FIG. 3 shows steps for constructing plasmid pAI234.

FIG. 4 shows steps for constructing plasmid pAI230.

FIG. 5 shows steps for constructing plasmid pAI282.

FIG. 6 shows steps for constructing plasmids pAI283 and pAI285.

FIG. 7 shows steps for constructing plasmids pAI284 and pAI289.

FIG. 8 shows steps for constructing plasmids pAI294 and pAI295.

FIG. 9 shows steps for constructing plasmids pAI299 and pAI301.

FIG. 10 shows steps for constructing plasmid pAI292.

FIG. 11 shows steps for constructing plasmid pAI297.

FIG. 12 shows steps for constructing plasmid pMKex1.

FIG. 13 shows steps for constructing plasmid pAI263.

FIG. 14 shows the binding reactivities of anti-human IL-5R α monoclonalantibody KM1257 and KM1259 with a human IL-5R α-human immunoglobulinconstant region fusion protein in an enzyme immunoassay.

FIG. 15 shows steps for constructing plasmid pBSA.

FIG. 16 shows steps for constructing plasmid pBSAE.

FIG. 17 shows steps for constructing plasmid pBSH-S.

FIG. 18 shows steps for constructing plasmid pBSK-H.

FIG. 19 shows steps for constructing plasmids pBSH-SA and pBSK-HA.

FIG. 20 shows steps for constructing plasmids pBSH-SAE and pBSK-HAE.

FIG. 21 shows steps for constructing plasmids pBSH-SAEE and pBSK-HAEE.

FIG. 22 shows steps for constructing plasmid pBSK-HAEESa1.

FIG. 23 shows steps for constructing plasmid pBSX-S.

FIG. 24 shows steps for constructing plasmid pBSX-SA.

FIG. 25 shows steps for constructing plasmid pBSSC.

FIG. 26 shows steps for constructing plasmid pBSMo.

FIG. 27 shows steps for constructing plasmid pBSMoS.

FIG. 28 shows steps for constructing plasmid pChiIgLA1S.

FIG. 29 shows steps for constructing plasmid pMohCK.

FIG. 30 shows steps for constructing plasmid pBSMoSa1.

FIG. 31 shows steps for constructing plasmid pBSMoSa1S.

FIG. 32 shows steps for constructing plasmid pBShCγ1.

FIG. 33 shows steps for constructing plasmid pMohCγ1.

FIG. 34 shows steps for constructing plasmid pMoγ1SP.

FIG. 35 shows steps for constructing plasmid pMoκγ1SP.

FIG. 36 shows steps for constructing plasmid pKANTEX93.

FIG. 37 shows steps for constructing plasmid pKANTEX1259H.

FIG. 38 shows steps for constructing plasmid pKANTEX1259.

FIG. 39 shows SDS-PAGE (on 4–15% gradient gel) electrophoresis patternsof anti-human IL-5R α chain human chimeric antibody KM1399. The left ofthe Figure shows the pattern of electrophoresis under non-reducingconditions and the right of the Figure under reducing conditions. On theleft-hand side, M is a lane of high molecular weight markers and 1 is alane of KM1399. On the right-hand side, M is a lane of low molecularweight markers and 1 is a lane of KM1399.

FIG. 40 shows the inhibition activities of anti-human IL-5R α chainmouse antibody KM1259 and anti-human IL-5R α chain human chimericantibody KM1399 against binding of human IL-5 to a human IL-5 α chain.The vertical axis of the graph plots the inhibition activity and thehorizontal axis, the antibody concentration. ● refers to the activity ofKM1259 and ∘, the activity of KM1399.

FIG. 41 shows steps for constructing plasmid pT1259.

FIG. 42 shows the results of evaluation of activity on the basis oftransient expression of an anti-human IL-5R α chain human chimericantibody using plasmid pT1259. The vertical axis of the graph plots theinhibition activity against binding of human IL-5 to a human IL-5R αchain and the horizontal axis plots the dilution factor for thetransient expression-culture supernatant.

FIG. 43 shows steps for constructing plasmid phKM1259HV0.

FIG. 44 shows steps for constructing plasmid phKM1259LV0.

FIG. 45 shows steps for constructing plasmid pKANTEX1259HV0.

FIG. 46 shows steps for constructing plasmid pKANTEX1259HV0LV0.

FIG. 47 shows SDS-PAGE (on 4–15% gradient gel) electrophoresis patternsof anti-human IL-5R α chain human CDR-grafted antibody KM8397. The leftof the Figure shows the pattern of electrophoresis under non-reducingconditions and the right of the Figure under reducing conditions. M is alane of molecular weight markers and 1 is a lane of KM8397.

FIG. 48 shows the activities of anti-human IL-5R α chain human chimericantibody KM1399 and anti-human IL-5R α chain human CDR-grafted antibodyKM8397 in binding to a human IL-5 α chain. The vertical axis of thegraph plots the activity in binding to the human IL-5 α chain and thehorizontal axis, an antibody concentration. ● refers to the activity ofKM1399 and ∘, the activity of KM8397.

FIG. 49 shows the results of evaluation of the activities of variousmodified versions of anti-human IL-5R α chain human CDR-graftedantibodies in transient expression-culture supernatants in inhibitingbinding of human IL-5 to a human IL-5 α chain. The vertical axis of thegraph plots the inhibitory activity and the horizontal axis indicatesthe names of samples. The inhibitory activity is expressed in relativeterms, with the activity of chimeric antibody KM1399 taken as 100.

FIG. 50 shows the activities of various modified versions of anti-humanIL-5R α chain human CDR-grafted antibodies in binding to a human IL-5 αchain. The vertical axis of each graph plots the activity in binding tothe human IL-5 α chain and the horizontal axis, the antibodyconcentration. In the upper graph, ● refers to the activity of KM1399;∘,HV.0LV.0; ▪, HV.2LV.0; □, HV.0LV.3; and ▴, HV.3LV.3. In the lower graph,● refers to the activity of KM1399;∘, HV.0LV.0; ▪, HV.3LV.0; □,HV.0LV.4; ▴, HV.1LV.4, Δ, HV.2LV.4; and x, HV.3LV.4.

FIG. 51 shows steps for constructing plasmid pBShCγ4.

FIG. 52 shows steps for constructing plasmids pKANTEX1259γ4 andpKANTEX1259HV3LV0γ4.

FIG. 53 shows SDS-PAGE (on 4–15% gradient gel) electrophoresis patternsof anti-human IL-5R α chain human chimeric antibody KM7399 of a humanantibody IgG4 subclass and human IL-5R α chain human CDR-graftedantibody KM9399 of a human antibody IgG4 subclass. The left of theFigure shows the pattern of electrophoresis under non-reducingconditions and the right of the Figure under reducing conditions. On theleft-hand side, M is a lane of high molecular weight markers, 1 is alane of KM9399 and 2 is a lane of KM7399. On the right-hand side, M is alane of low molecular weight markers, 1 is a lane of KM9399 and 2 is alane of KM7399.

FIG. 54 shows the activity of anti-human IL-5R α chain human chimericantibody KM1399 of a human antibody IgG1 subclass, human IL-5R α chainhuman chimeric antibody KM7399 of a human antibody IgG4 subclass,anti-human IL-5R α chain human CDR-grafted antibody KM8399 of a humanantibody IgG1 subclass and anti-human IL-5R α chain human CDR-graftedantibody KM9399 of a human antibody IgG4 subclass in binding to a humanIL-5 α chain. The vertical axis of the graph plots the activity ofbinding to the human IL-5 α chain and the horizontal axis, the antibodyconcentration. ∘ refers to the activity of KM1399;●, KM7399; □, KM8399;and ▪, KM9399.

FIG. 55 shows the results of flowcytometric analysis of the reactivitiesof anti-human IL-5R α monoclonal antibodies KM1257, KM1259, KM1486,KM1399, KM7399, KM8399 and KM9399 with a human IL-5R gene-transfectedCTLL-2 cell.

FIG. 56 shows the results of examination of the inhibitory action ofanti-human IL-5R α monoclonal antibodies KM1257, KM1259, KM1486, KM1399,KM7399, KM8399 and KM9399 against IL-5-dependent growth of a human IL-5Rgene-transfected CTLL-2 cell.

FIG. 57 shows the results of flowcytometric analysis of the reactivityof anti-human IL-5R α monoclonal antibody KM1259 with human eosinophils.

FIG. 58 shows the results of examination of inhibitory action ofanti-human IL-5R α monoclonal antibodies KM1257, KM1259, KM1486, KM1399, KM7399, KM8399 and KM9399 for the survival of human eosinophils.

FIG. 59 shows the results of evaluation of a soluble human IL-5R αquantitative determination system using anti-human IL-5R α monoclonalantibody KM1257 and biotin-labeled KM1259.

FIG. 60 shows the results of detection of shIL-5R α by Western blottingusing anti-human IL-5R α monoclonal antibodies KM1257, KM1259 andKM1486.

FIG. 61 shows the results of immunoprecipitation of shIL-5R α usinganti-human IL-5R a monoclonal antibodies KM1257, KM1259 and KM1486.

EXAMPLES Example 1

1. Preparation of Antigens

(1) Construction Expression Vector for Animal Cell pAGE210

Expression vector for animal cell, pAGE210, was constructed as describedbelow using expression vectors for animal cell pAGE207 (Kokai No.46841/94) and pAGE148 (Kokai No. 205694/94).

Three μg of plasmid pAGE207 or pAGE148 was dissolved in 30 μl of abuffer containing 10 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride, 50mM sodium chloride and 1 mM dithiothreitol (hereinafter referred to as“DTT”). To the resultant mixture, 10 units each of ClaI and KpnI (bothmanufactured by Takara Shuzo; unless otherwise indicated, therestriction enzymes used herein below are those manufactured by TakaraShuzo) were added and reacted at 37° C. for 4 hours. After the reactionmixture was subjected to agarose gel electrophoresis, about 0.5 μg of a4.7 kb DNA fragment containing the SV40 early promoter and enhancer(hereinafter referred to as “P_(SE)”), a hygromycin resistance gene andan ampicillin resistance gene was recovered from pAGE207 and about 0.5%gof a 4.3 kb DNA fragment containing a dihydrofolate reductase(hereinafter referred to as “dhfr”) gene was recovered from pAGE148.

The ClaI-KpnI fragment obtained from pAGE207 (50 ng) and the KpnI-ClaIfragment obtained from pAGE148 (50 ng) were dissolved in 20 μl of T4DNAligase buffer [a buffer containing 66 mM Tris-HCl (pH 7.5), 6.6 mMmagnesium chloride, 10 mM DTT and 0.1 mM adenosine triphosphate(hereinafter referred to as “ATP”]. To the resultant mixture, 200 unitsof T4DNA ligase (Takara Shuzo) was added and ligation was performed at12° C. for 16 hours. Using the prepared recombinant plasmid DNA, E. colistrain JM109 was transformed to thereby obtain plasmid pAGE210 shown inFIG. 1.

(2) Making shIL-5R α cDNA into a Cassette for the Construction of anshIL-5R α Expression Vector

In order to construct an shIL-5R α expression vector, the modificationof the 5′ and 3′ non-translational region of shIL-5R α cDNA and theintroduction of a restriction enzyme recognition sequence were carriedout using the PCR method [Maniatis et al. (eds.), MolecularCloning,-14.2, Cold Spring Harbor Laboratory, 1989] according to theprocedures described below.

Plasmid pCAGGS-h5R.25 is obtained by inserting shIL-5R α cDNA into theknown plasmid pCAGGS [Gene, 108, 193 (1991)] as shown in FIG. 2 [J. Exp.Med., 175, 341 (1992)]. Three μg of this pCAGGS-h5R.25 were added to 30μL of a buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM magnesiumchloride, 100 mM sodium chloride and 1 mM DTT. Then, 10 units of EcoRIwere added thereto, and reacted at 37° C. for 4 hours. After thereaction mixture was subjected to agarose gel electrophoresis, about 0.3μg of a 1.4 kb DNA fragment containing shIL-5R α cDNA was recovered.

Then, 1 ng of the DNA fragment obtained above was dissolved in 50 μl ofPCR buffer [a buffer containing 50 mM potassium chloride, 10 mM Tris-HCl(pH 8.3), 1.5 mM magnesium chloride, 0.2 mM deoxyadenosine triphosphate(hereinafter referred to as “dATP”), 0.2 mM deoxyguanosine triphosphate(hereinafter referred to as “dGTP”), 0.2 mM deoxycytosine triphosphate(hereinafter referred to as “dCTP”) and 0.2 mM deoxythymidinetriphosphate (hereinafter referred to as “dTTP”)]. To the resultantmixture, 50 pmol each of a synthetic DNA having the base sequence shownin SEQ ID NO: 1 and a synthetic DNA having the base sequence shown inSEQ ID NO: 2 [both synthesized with an automatic DNA synthesizer; Model380A (Applied Biosystems Co., Ltd.)] and 1.6 units of Vent DNApolymerase (New England BioLabs, Inc.) were added and PCR was performedthrough 30 cycles under a series of conditions of 94° C. for 1 minute,55° C. for 2 minutes and 72° C. for 3 minutes using a Perkin Elmer DNAthermal cycler (this was also used for the other PCR reactions). Afterthe completion of the reaction, 2 μl of a buffer containing 100 mMTris-HCl (pH 7.5), 100 mM magnesium chloride, 500 mM sodium chloride and10 mM DTT, 8 μl of distilled water, and 10 units of HindIII were addedto 10 μl of the reaction mixture and reacted at 37° C. for 4 hours.Then, DNA fragments were recovered from the reaction mixture by ethanolprecipitation [Maniatis et al. (eds.), Molecular Cloning, E.10, ColdSpring Harbor Laboratory, 1989] and redissolved in 20 μl of a buffercontaining 20 mM Tris-HCl (pH 8.5), 10 mM magnesium chloride, 10 mMpotassium chloride and 1 mM DTT. To the resultant mixture, 10 units ofBamHI were added and reacted at 37° C. for 4 hours. After the reactionmixture was subjected to agarose gel electrophoresis, about 0.3 μg of a1.0 kb DNA fragment was recovered.

In a separate step, 3 μg of plasmid pUC19 (Pharmacia Biotech) wasdissolved in 30 μl of a buffer containing 10 mM Tris-HCl (pH 7.5), 10 mMmagnesium chloride, 50 mM sodium chloride and 1 mM DTT, to which 10units of HindIII were added and reacted at 37° C. for 4 hours.Thereafter, DNA fragments were recovered from the reaction mixture byethanol precipitation and redissolved in 30 μl of a buffer containing 20mM Tris-HCl (pH 8.5), 10 mM magnesium chloride, 10 mM potassium chlorideand 1 mM DTT. To the resultant mixture, 10 units of BamHI were added andreacted at 37° C. for 4 hours. After the reaction mixture was subjectedto agarose gel electrophoresis, about 0.5 μg of the HindIII/BamHIfragment from pUC 19 was recovered.

One hundred ng of the HindIII/BamHI fragment from pUC19 and 50 ng ofshIL-5R α cDNA fragment were dissolved in 20 μl of T4DNA ligase buffer,to which 200 units of T4DNA ligase were added. Then, ligation wasperformed at 12° C. for 16 hours. Using the recombinant plasmid DNA thusprepared, E. coli strain JM109 was transformed to thereby obtain plasmidpAI234 shown in FIG. 3.

(3) Construction of a Human Soluble IL-5R α Expression Vector

An shIL-5R α expression vector, pAI230, was constructed as describedbelow by ligating the HindIII-BamHI fragment from pAGE210 obtained insubsection (1) of Example 1 to the shIL-5R α cDNA-containingHindIII-BamHI fragment from pAI234 obtained in subsection (2) of Example1.

Briefly, 3 μg of pAGE210 was added to 30 μl of a buffer containing 10 mMTris-HCl (pH 7.5), 10 mM magnesium chloride, 50 mM sodium chloride and 1mM DTT, to which 10 units of HindIII were added and reacted at 37° C.for 4 hours. DNA fragments were recovered from the reaction mixture byethanol precipitation and redissolved in 30 μl of a buffer containing 20mM Tris-HCl (pH 8.5), 10 mM magnesium chloride, 10 mM potassium chlorideand 1 mM DTT, to which 10 units of BamHI were added and reacted at 37°C. for 4 hours. After the reaction mixture was subjected to agarose gelelectrophoresis, about 0.5 μg of a 9.0 kb DNA fragment was recovered.

Three μg of pAI234 were added to 30 μl of a buffer containing 10 mMTris-HCl (pH 7.5), 10 mM magnesium chloride, 50 mM sodium chloride and 1mM DTT, to which 10 units of HindIII were added and reacted at 37° C.for 4 hours. DNA fragments were recovered from the reaction mixture byethanol precipitation and redissolved in 30 μl of a buffer containing 20mM Tris-HCl (pH 8.5), 10 mM magnesium chloride, 10 mM potassium chlorideand 1 mM DTT, to which 10 units of BamHI was added and reacted at 37° C.for 4 hours. After the reaction mixture was subjected to agarose gelelectrophoresis, about 0.3 μg of a 1.0 kb DNA fragment was recovered.

Subsequently, 300 ng of the HindIII-BamHI fragment from pAGE210 and 50ng of the HindIII-BamHI fragment from pAI234 were dissolved in 20 μl ofT4DNA ligase buffer, to which 200 units of T4DNA ligase were added.Then, ligation was performed at 12° C. for 16 hours. Using therecombinant plasmid DNA thus prepared, E. coli strain JM109 wastransformed to thereby obtain plasmid pAI230 shown in FIG. 4.

(4) Modification of the Signal Sequence

In order to produce shIL-5R α efficiently in animal cells, the signalsequence of the cDNA coding for shIL-5R α was modified according to theprocedures described below by introducing an EcoRV recognition sequenceinto the cDNA at the 3′ end of the signal sequence and subsequentlyreplacing the original signal sequence with a signal sequence from ahuman growth hormone [Science, 205, 602 (1979)] or anti-ganglioside GD3chimeric antibody KM871 (Kokai No. Hei 5-304989) using synthetic DNAs.Briefly, 3 μg of plasmid pAI234 obtained in subsection (2) of Example 1were added to 30 μl of a buffer containing 10 mM Tris-HCl (pH 7.5), 10mM magnesium chloride, 50 mM sodium chloride and 1 mM DTT, to which 10units of HindIII were added and reacted at 37° C. for 4 hours. DNAfragments were recovered from the reaction mixture by ethanolprecipitation and redissolved in 30 μl of a buffer containing 20 mMTris-HCl (pH 8.5), 10 mM magnesium chloride, 10 mM potassium chlorideand 1 mM DTT, to which 10 units of BamHI were added and reacted at 37°C. for 4 hours. After the reaction mixture was subjected to agarose gelelectrophoresis, about 0.3 μg of a 1.0 kb DNA fragment was recovered.

In a separate step, 3 μg of plasmid pUC19 were added to 30 μl of abuffer containing 10 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride, 50mM sodium chloride and 1 mM DTT, to which 10 units of HincII were addedand reacted at 37° C. for 4 hours. Then, DNA fragments were recoveredfrom the reaction mixture by ethanol precipitation and about 0.5 μg of aHincII fragment from pUC19 was recovered.

About 1 ng of the DNA fragment obtained above was dissolved in 50 μl ofPCR buffer, to which 50 pmol each of a synthetic DNA having the basesequence shown in SEQ ID NO: 2 and a synthetic DNA having the basesequence shown in SEQ ID NO: 3 and 1.6 units of vent DNA polymerase wereadded. Then, PCR was performed through 30 cycles under a series ofconditions of 94° C. for 1 minute, 48° C. for 2 minutes and 72° C. for 3minutes. Then, the reaction mixture was subjected to agarose gelelectrophoresis, and 0.5 μg of about 0.9 kb cDNA fragment coding for aportion of hIL-5R α was recovered. Fifty ng of this DNA and 100 ng ofthe HincII fragment from pUC19 were dissolved in 20 μl of T4 ligasebuffer, to which 200 units of T4DNA ligase were added. Then, ligationwas performed at 12° C. for 16 hours. Using the recombinant plasmid DNAthus prepared, E. coli strain JM109 was transformed to thereby obtainplasmid pAI280 shown in FIG. 5. Three μg of the thus obtained plasmidpAI280 were added to 30 μl of a buffer containing 10 mM Tris-HCl (pH7.5), 10 mM magnesium chloride, 50 mM sodium chloride and 1 mM DTT, towhich 10 units of XbaI were added and reacted at 37° C. for 4 hours. DNAfragments were recovered from the reaction mixture by ethanolprecipitation and redissolved in 30 μl of a buffer containing 20 mMTris-HCl (pH 8.5), 10 mM magnesium chloride, 10 mM potassium chlorideand 1 mM DTT, to which 10 units of BamHI were added and reacted at 37°C. for 4 hours. After the reaction mixture was subjected to agarose gelelectrophoresis, about 0.8 μg of a 2.8 kb DNA fragment was recovered.

In a separate step, 3 μg of plasmid pAI234 were added to 30 μl of abuffer containing 10 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride, 50mM sodium chloride and 1 mM DTT, to which 10 units of XbaI were addedand reacted at 37° C. for 4 hours. DNA fragments were recovered from thereaction mixture by ethanol precipitation and redissolved in 30 μl of abuffer containing 20 mM Tris-HCl (pH 8.5), 10 mM magnesium chloride, 10mM potassium chloride and 1 mM DTT, to which 10 units of BamHI wereadded and reacted at 37° C. for 4 hours. After the reaction mixture wassubjected to agarose gel electrophoresis, about 0.2 μg of a 0.8 kb DNAfragment was recovered.

Subsequently, 200 ng of the XbaI-BamHI from pAI280 and 50 ng of theXbaI-BamHI from pAI234 were dissolved in 20 μl of T4 ligase buffer, towhich 200 units of T4DNA ligase were added. Then, ligation was performedat 12° C. for 16 hours. Using the recombinant plasmid DNA thus prepared,E. coli strain JM109 was transformed to thereby obtain plasmid pAI282shown in FIG. 5. Three μg of this plasmid pAI282 were added to 30 μl ofa buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride,100 mM sodium chloride and 1 mM DTT, to which 10 units of EcoRV wereadded and reacted at 37° C. for 4 hours. DNA fragments were recoveredfrom the reaction mixture by ethanol precipitation and redissolved in 30μl of a buffer containing 20 mM Tris-HCl (pH 8.5), 10 mM magnesiumchloride, 10 mM potassium chloride and 1 mM DTT, to which 10 units ofBamHI were added and reacted at 37° C. for 4 hours. After the reactionmixture was subjected to agarose gel electrophoresis, about 0.3 μg of a0.9 kb DNA fragment was recovered.

One μg each of a synthetic DNA having the base sequence shown in SEQ IDNO: 4 and a synthetic DNA having the base sequence shown in SEQ ID NO: 5were dissolved in 10 μl of distilled water. The resultant mixture washeated at 95° C. for 5 minutes and then cooled to room temperature over30 minutes for annealing. A hundred ng of the HindIII-BamHI fragmentfrom pUC19 obtained in subsection (2) of Example 1, 50 ng of theEcoRV-BamHI fragment from pAI282, and 50 ng of the synthetic DNAs havingthe base sequences shown in SEQ ID NOS. 4 and 5 which had been annealedas described above were dissolved in 20 μl of T4DNA ligase buffer, towhich 200 units of T4DNA ligase was added. Then, ligation was performedat 12° C. for 16 hours. Using the recombinant plasmid DNA thus prepared,E. coli strain JM109 was transformed to thereby obtain plasmid pAI283shown in FIG. 6.

One μg each of a synthetic DNA having the base sequence shown in SEQ IDNO: 6 and a synthetic DNA having the base sequence shown in SEQ ID NO: 9were dissolved in 10 μl of distilled water. The resultant mixture washeated at 95° C. for 5 minutes and then cooled to room temperature over30 minutes for annealing. To this reaction mixture, 2.5 μl of a buffercontaining 500 mM Tris-HCl (pH 7.6), 100 mM magnesium chloride, 50 mMDTT and 1 mM EDTA, 2.5 μl of 10 mM ATP solution, 9 μl of distilled waterand 5 units of T4 polynucleotide kinase (Takara Shuzo) were added, andphosporylation was performed at 37° C. for 2 hours. Separately, 1 μgeach of a synthetic DNA having the base sequence shown in SEQ ID NO: 7and a synthetic DNA having the base sequence shown in SEQ ID NO: 8 weredissolved in 10 μl of distilled water. The resultant mixture was heatedat 95° C. for 5 minutes and then cooled to room temperature over 30minutes for annealing.

One hundred ng of the HindIII-BamHI fragment from pUC19, 50 ng of theEcoRV-BamHI fragment from pAI282, and 50 ng each of the synthetic DNAsas prepared above were dissolved in 20 μl of T4DNA ligase buffer, towhich 200 units of T4DNA ligase were added. Then, ligation was performedat 12° C. for 16 hours. Using the thus prepared recombinant plasmid DNA,E. coli strain JM109 was transformed to thereby obtain plasmid pAI285shown in FIG. 6.

(5) Construction of Signal Sequence-Modified shIL-5R α ExpressionVectors

Human soluble IL-5R α expression vectors, pAI284 and pAI289, wereconstructed as described below by ligating the HindIII-BamHI fragmentfrom pAGE210 obtained in subsection (1) of Example 1 to theHindIII-BamHI fragment containing human soluble IL-5R α cDNA from pAI283or pAI285 obtained in subsection (4) of Example 1.

Briefly, 3 μg each of pAI283 and pAI285 were added separately to 30 μlof a buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM magnesiumchloride, 50 mM sodium chloride and 1 mM DTT, to which 10 units ofHindIII were added and reacted at 37° C. for 4 hours. DNA fragments wererecovered from the reaction mixture by ethanol precipitation andredissolved in 30 μl of a buffer containing 20 mM Tris-HCl (pH 8.5), 10mM magnesium chloride, 10 mM potassium chloride and 1 mM DTT, to which10 units of BamHI were added and reacted at 37° C. for 4 hours. Afterthe reaction mixture was subjected to agarose gel electrophoresis, about0.3 μg of a 1.0 kb DNA fragment was recovered for each of the plasmidsused.

Three hundred ng of the HindIII-BamHI fragment from pAGE210 and 50 ng ofthe HindIII-BamHI fragment from pAI283 or pAI285 were dissolved in 20 μlof T4DNA ligase buffer, to which 200 units of T4DNA ligase were added.Then, ligation was performed at 12° C. for 16 hours. Using therecombinant plasmid DNA thus prepared, E. coli strain JM109 wastransformed to thereby obtain plasmids pAI284 and pAI289 shown in FIG.7.

(6) Preparation of a Fusion Protein Composed of Human IL-5R α and HumanImmunoglobulin Constant Region

A fusion protein in which the extracellular region of human IL-5R α waslinked to a human immunoglobulin constant region (hereinafter referredto as “Fc”) through a linker having an amino acid sequence of(Gly-Ser-Gly)₄ (hereinafter, this fusion protein is referred to as“hIL-5R α-Fc”) was prepared according to the procedures described below.

As a cDNA coding for a human immunoglobulin constant region, the portionof the human chimeric antibody H chain expression vector pChiIgHB2(Kokai No. Hei 5-304989) which coded for the human IgG1 constant regionwas used. First, about 1 ng of pChilgHB2 was dissolved in 50 μl of PCRbuffer. To this solution, 50 pmol each of a synthetic DNA having thebase sequence shown in SEQ ID NO: 10 and a synthetic DNA having the basesequence shown in SEQ ID NO: 11 and 1.6 units of vent DNA polymerasewere added. Then, PCR was performed through 30 cycles under a series ofconditions of 94° C. for 1 minute, 48° C. for 2 minutes and 72° C. for 3minutes. After the completion of the reaction, 2.5 μl of a buffercontaining 200 mM Tris-HCl (pH 8.5), 100 mM magnesium chloride, 1000 mMpotassium chloride and 10 mM, 2.5 μl of distilled water, and 10 units ofBamHI were added to 20 μl of the reaction mixture and reacted at 37° C.for 4 hours. After the completion of the reaction, the reaction mixturewas subjected to agarose gel electrophoresis, and about 0.5 μg of a 0.7kb DNA fragment containing a cDNA coding for the human IgG1 constantregion was recovered.

About 1 ng of pAI283 obtained in subsection (4) of Example 1 wasdissolved in 50 μl of PCR buffer, to which 50 pmol each of a syntheticDNA having the base sequence shown in SEQ ID NO: 12 and a synthetic DNAhaving the base sequence shown in SEQ ID NO: 13 and 1.6 units of ventDNA polymerase were added. Then, PCR was performed through 30 cyclesunder a series of conditions of 94° C. for 1 minute, 48° C. for 2minutes and 72° C. for 3 minutes. After the completion of the reaction,2.5 μl of a buffer containing 100 mM Tris-HCl (pH 7.5), 100 mM magnesiumchloride, 500 mM sodium chloride and 10 mM DTT, 2.5 μl of distilledwater, and 10 units of HindIII were added to 20 μl of the reactionmixture and reacted at 37° C. for 4 hours. After the completion of thereaction, the reaction mixture was subjected to agarose gelelectrophoresis. Thereafter, about 0.5 μg of a 1.0 kb DNA fragmentcontaining a cDNA coding for the extracellular region of hIL-5R α wasrecovered.

Fifty ng of the 0.7 kb DNA fragment containing the cDNA coding for thehuman IgG1 constant region, 50 ng of the DNA fragment containing thecDNA coding for the extracellular region of hIL-5R α and 100 ng of theHindIII-BamHI fragment from pUC 19 were dissolved in 20 μl of T4DNAligase buffer, to which 200 units of T4DNA ligase were added. Then,ligation was performed at 12° C. for 16 hours. Using the recombinantplasmid DNA thus prepared, E. coli strain JM109 was transformed tothereby obtain plasmid pAI294 shown in FIG. 8.

In a separate step, PCR reaction was conducted under conditions similarto those described above using pAI285 obtained in subsection (4) ofExample 1 as a template and also using synthetic DNAs having the basesequences shown in SEQ ID NOS: 13 and 14, as primers. After thecompletion of the reaction, the reaction mixture was subjected toagarose gel electrophoresis. Subsequently, about 0.5 μg of a 1.0 kb DNAfragment containing the cDNA coding for the extracellular region ofhuman IL-5R α was recovered. Fifty ng of the thus obtained DNA fragment,50 ng of the 0.7 kb DNA fragment containing the cDNA coding for thehuman IgG1 constant region and 100 ng of the HindIII-BamHI fragment frompUC19 were dissolved in 20 μl of T4DNA ligase buffer, to which 200 unitsof T4DNA ligase were added. Then, ligation was performed at 12° C. for16 hours. Using the recombinant plasmid DNA thus prepared, E. colistrain JM109 was transformed to thereby obtain plasmid pAI295 shown inFIG. 8.

(7) Construction of a Fusion Protein Expression Vector

An hIL-5R α-Fc expression vector, pAI299, was constructed as describedbelow by ligating the HindIII-BamHI fragment from pAGE210 obtained insubsection (1) of Example 1 to the HindIII-BamHI fragment from pAI294obtained in subsection (6) of Example 1 containing the cDNA coding forhIL-5R α-Fc.

Briefly, 3 μg of plasmid pAI294 were added to 30 μl of a buffercontaining 10 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride, 50 mMsodium chloride and 1 mM DTT, to which 10 units of HindIII were addedand reacted at 37° C. for 4 hours. DNA fragments were recovered from thereaction mixture by ethanol precipitation and redissolved in 30 μl of abuffer containing 20 mM Tris-HCl (pH 8.5), 10 mM magnesium chloride, 100mM potassium chloride and 1 mM DTT. To the resultant mixture, 10 unitsof BamHI were added and reacted at 37° C. for 4 hours. After thereaction mixture was subjected to agarose gel electrophoresis, about 0.4μg of a 1.7 kb DNA fragment containing a cDNA coding for a fusionprotein composed of human IL-5R α and the human immunoglobulin constantregion was recovered.

One hundred ng of the HindIII-BamHI fragment from pAGE210 and 50 ng ofthe HindIII-BamHI fragment from pAI294 were dissolved in 20 μl of T4DNAligase buffer, to which 200 units of T4DNA ligase were added. Then,ligation was performed at 12° C. for 16 hours. Using the recombinantplasmid DNA thus prepared, E. coli strain JM109 was transformed tothereby obtain plasmid pAI299 shown in FIG. 9.

Further, an hIL-5R α-Fc expression vector, pAI301, was constructedsimilarly by ligating the HindIII-BamHI fragment from pAGE210 to theHindIII-BamHI fragment from pAI295 obtained in subsection (6) of Example1 containing the cDNA coding for hIL-5R α-Fc.

(8) Preparation of a Recombinant Virus for Expressing shIL-5R α inInsect Cells

For the production of a protein in insect cells, a recombinant virusinserting a gene of interest is prepared. The preparation of such avirus is performed through a process in which a cDNA coding for a geneof interest is incorporated into a special plasmid called “a transfervector” and a subsequent process in which a wild-type virus and thetransfer vector are co-transfected into insect cells to obtain arecombinant virus by homologous recombination. The processes describedabove were performed using BaculoGold Starter Kit (Cat. No. PM-21001K)manufactured by Pharmingen according to the manufacturer's manual.

Briefly, 3 μg of pAI285 obtained in subsection (4) of Example 1 orpAI294 obtained in subsection (6) of Example 1 were added to 30 μL of abuffer containing 10 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride, 50mM sodium chloride and 1 mM DTT, to which 10 units of HindIII were addedand reacted at 37° C. for 4 hours. DNA fragments were recovered from thereaction mixture by ethanol precipitation and dissolved in 20 μl of DNApolymerase I buffer [a buffer containing 5 mM Tris-HCl (pH 7.5), 1 mMmagnesium sulfate, 0.01 mM DTT, 5 μg/ml bovine serum albumin, 0.08 mMdATP, 0.08 mM dGTP, 0.08 mM dCTP and 0.08 mM dTTP]. To the resultantmixture, 5 units of E. coli DNA polymerase I Klenow fragment (TakaraShuzo) were added and reacted at 22° C. for 30 minutes, whereby the 5′sticky ends generated by the HindIII digestion were changed to bluntends. Further, the reaction mixture was subjected to phenol-chloroformextraction followed by ethanol precipitation. To the precipitate, 30 μlof a buffer containing 20 mM Tris-HCl (pH 8.5), 10 mM magnesiumchloride, 100 mM potassium chloride and 1 mM DTT, and 10 units of BamHIwere added and reacted at 37° C. for 4 hours. The reaction mixture wassubjected to agarose gel electrophoresis, and about 0.3 μg of an approx.1.0 kb DNA fragment containing the cDNA coding for shIL-5R α and about0.3 μg of a 1.7 kb DNA fragment containing the cDNA coding for thefusion protein composed of human IL-5 α and the human immunoglobulinconstant region were recovered. Subsequently, 3 μg of plasmid pVL1393contained in BaculoGold Starter Kit (Pharmingen) were added to 30 μl ofa buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride,100 mM sodium chloride and 1 mM DTT, to which 10 units of EcoRI wereadded and reacted at 37° C. for 4 hours. DNA fragments were recoveredfrom the reaction mixture by ethanol precipitation and dissolved in 20μl of DNA polymerase I buffer, to which 5 units of E. coli DNApolymerase I Klenow fragment were added and reacted at 22° C. for 30minutes, whereby the 5′ sticky ends generated by the EcoRI digestionwere changed to blunt ends. Further, the reaction mixture was subjectedto phenol-chloroform extraction followed by ethanol precipitation. Tothe precipitate, 30 μl of a buffer containing 50 mM Tris-HCl (pH 7.5),10 mM magnesium chloride, 100 mM sodium chloride and 1 mM DTT, and 10units of BglII were added and reacted at 37° C. for 4 hours. Thereaction mixture was subjected to agarose gel electrophoresis, and about0.9 μg of an approx. 9.6 kb DNA fragment was recovered.

Thereafter, 200 ng of the thus obtained EcoRI (blunt end)-BglII fragmentfrom pVL1393 and 50 ng of the HindIII (blunt end)-BamHI fragment frompAI285 or pAI294 were dissolved in 20 μl of T4DNA ligase buffer, towhich 200 units of T4DNA ligase were added. Then, ligation was performedat 12° C. for 16 hours. Using the recombinant plasmid DNA thus prepared,E. coli strain JM109 was transformed to thereby obtain plasmids pAI292and pAI297 shown in FIGS. 10 and 11, respectively.

The subsequent preparation of a recombinant virus was performed asdescribed below by transfecting into an insect cell, Sf9 (obtained fromPharmingen), cultured in TMN-FH Insect Medium (Pharmingen), a linearbaculovirus DNA (BaculoGold baculovirus DNA; Pharmingen) and theprepared transfer vector DNA by the lipofectin method [TANPAKUSHITSU,KAKUSAN, KOHSO (Protein, Nucleic Acid, Enzyme), 37, 2701 (1992)].

Briefly, 1 μg of pAI292 or pAI297 and 20 ng of the linear baculovirusDNA were dissolved in 12 μl of distilled water, to which a mixture of 6μl of lipofectin and 6 μl of distilled water were added and left at roomtemperature for 15 minutes. In a separate step, 1×10⁶ Sf9 cells weresuspended in 2 ml of Sf900-II medium (Gibco) and put in a plastic cellculture dish 35 mm in diameter. To this dish, a total volume of theabove-described mixture of plasmid DNA, linear baculovirus DNA andlipofectin was added, and cells were cultured at 27° C. for 3 days.Thereafter, 1 ml of the culture supernatant containing a recombinantvirus was taken. One ml of a fresh Sf900-II medium was added to the dishand cells were cultured at 27° C. for another 3 days. Then, anadditional 1.5 ml of the culture supernatant containing a recombinantvirus was obtained.

Subsequently, the thus obtained recombinant virus was propagated for thepurpose of use in protein expression, according to the proceduresdescribed below.

Briefly, 2×10⁷ Sf9 cells were suspended in 10 ml of Sf900-II medium, putin a 175 cm2 flask (Greiner) and left at room temperature for 1 hour toallow cells to adhere to the flask. Thereafter, the supernatant wasremoved, and 15 ml of a fresh TMN-FH Insect Medium and 1 ml of theabove-obtained culture supernatant containing the recombinant virus wereadded to the flask. Then, cells were cultured at 27° C. for 3 days.After the cultivation, the supernatant was centrifuged at 1,500×g for 10minutes to remove cells. Thus, a viral solution to be used for proteinexpression was obtained.

With respect to the thus obtained solution of the recombinant virus, theviral titer was calculated by the method described below (BaculoGoldStarter Kit Manual; Pharmingen). A number (6×10⁶) of Sf9 cells weresuspended in 4 ml of Sf900-II medium, put in a plastic cell culture dish60 mm in diameter and left at room temperature for 1 hour to allow cellsto adhere to the dish. After the removal of the supernatant, 400 μl of afresh Sf900-II medium and the above-described recombinant virus solutiondiluted 10,000 folds with Sf900-II medium were added to the dish andleft at room temperature for 1 hour. Then, the medium was removed, and 5ml of a medium containing 1% low melting point agarose (AgarplaqueAgarose; Pharmingen) (a medium obtainable by mixing 1 ml of sterilized5% aqueous Agarplaqueplus Agarose solution and 4 ml of TMN-FH InsectMedium and keeping the mixture at 42° C.) was poured into the dish.After the dish was left at room temperature for 15 minutes, vinyl tapewas wound round the dish to prevent dryness. Then, the dish was placedin an airtight plastic container and cells were cultured at 27° C. for 6days. After 1 ml of PBS containing 0.01% Neutral Red was added to thedish and cells were cultured for an additional day, the number ofplaques formed was counted. From the operations described above, it wasfound that each of the recombinant virus solutions contained about 1×10⁷plaque forming units(PFU)/ml of virus.

(9) Expression of shIL-5R α or hIL-5R α-Fc in Animal Cells

The introduction of a plasmid into animal cells was performed accordingto the method of Miyaji et al. using electroporation [Cytotechnology, 3,133 (1990)].

Briefly, 4 μg of pAI289 obtained in subsection (5) of Example 1 orpAI301 obtained in subsection (7) of Example 1 were transfected into4×10⁶ dhfr gene-deficient CHO cells [Proc. Natl. Acad. Sci., 77, 4216(1980)], which were then suspended in 40 ml of RPMI1640-FCS(10)[RPMI1640 medium containing 10% FCS, 1/40 volume 7.5% NaHCO3, 3% 200 mML-glutamine solution (Gibco) and 0.5% penicillin/streptomycin solution(Gibco; containing 5000 units/ml penicillin and 5000 μg/mlstreptomycin); manufactured by Nissui Pharmaceuticals] and dispensedinto a 96-well microtiter plate (200 μl/well). After the cells werecultured in a CO₂ incubator at 37° C. for 24 hours, hygromycin (Gibco)was added to give a concentration of 0.5 mg/ml. Then, the cells werecultured for an additional 1–2 weeks. Cells were recovered from thosewells which became confluent with the appearance of colonies oftransformant, and suspended in RPMI1640-FCS(10) medium containing 0.5mg/ml hygromycin and 50 nM methotrexate (hereinafter referred to as“MTX”) to give a cell density of 1–2×10⁵ cells/ml. The cell suspensionwas dispensed into a 24-well plate (2 ml/well) and the cells werecultured in a CO₂ incubator at 37° C. for 1–2 weeks to thereby induce 50nM MTX resistant clones.

The thus obtained 50 nM MTX resistant clones were suspended inRPMI1640-FCS(10) medium containing 0.5 mg/ml hygromycin and 200 nM MTXto give a cell density of 1–2×10⁵ cells/ml. The cell suspension wasdispensed into a 24-well plate (2 ml/well) and the cells were culturedin a CO₂ incubator at 37° C. for 1–2 weeks to thereby induce 200 nM MTXresistant clones.

Further, the thus obtained 200 nM MTX resistant clones were suspended inRPMI1640-FCS(10) medium containing 0.5 mg/ml hygromycin and 500 nM MTXto give a cell density of 1–2×10⁵ cells/ml. The cell suspension wasdispensed into a 24-well plate (2 ml/well) and the cells were culturedin a CO₂ incubator at 37° C. for 1–2 weeks to thereby induce 500 nM MTXresistant clones.

The above transformants were suspended in a serum-free medium for CHOcells, CHO-S-SFMII medium (Gibco), to give a cell density of 1–2×10⁵cells/ml, and the cell suspension was dispensed into 225 cm2 flasks(Greiner) in an amount of 100 ml/flask. The cells were cultured in a CO₂incubator at 37° C. for 5–7 days and the culture medium was recoveredwhen confluence was attained.

The purification of hIL-5R α from the culture supernatant was performedas follows. To 1 liter of the culture medium of pAI289-derivedtransformant, 29.2 g of sodium chloride and 20 ml of 1 M Tris-HCl (pH7.4) were added. Then, the pH of the resultant mixture was adjusted to7.4 with 1 N sodium hydroxide solution. A column was packed with about10 ml of Concanavalin A-Sepharose (Pharmacia) gel and then washed with50 ml of a buffer containing 20 mM Tris-HCl (pH 7.4) and 0.5 M sodiumchloride at a flow rate of 0.5 ml/min. After the washing, the mixturecontaining shIL-5R α prepared as described above was applied to theConcanavalin A-Sepharose column at a flow rate of 0.5 ml/min. Then, thecolumn was washed with 80 ml of a buffer containing 20 mM Tris-HCl (pH7.4) and 0.5 M sodium chloride at a flow rate of 0.5 ml/min. Thereafter,the protein adsorbed on Concanavalin A-Sepharose was eluted and,simultaneously, the eluate was fractionated into 1 ml fractions(fractions 1–30) with 15 ml of a buffer containing 20 mM Tris-HCl (pH7.4) and 0.5 M sodium chloride and 15 ml of a buffer containing 0.5 Mα-methylmannoside, 20 mM Tris-HCl (pH 7.4) and 0.5 M sodium chloride bylinearly changing the a-methylmannoside concentration from 0 to 0.5 M.Further, 20 ml of a buffer containing 1 M α-methylmannoside, 20 mMTris-HCl (pH 7.4) and 0.5 M sodium chloride were applied to the columnand the eluate was fractionated into 2 ml fractions (fractions 31–40).The protein concentration of each fraction was measured using a proteinconcentration measurement kit (Bio-rad) and fractions 10–40 having highprotein concentration were recovered. The resultant protein solution wasconcentrated by a factor of about 10 using Centricon-30 (Amicon), placedin a dialysis tube and dialyzed against PBS. Thus, a purified shIL-5R α(protein concentration: 4 mg/ml; 3.5 ml) was obtained.

In a separate step, hIL-5R α-Fc was obtained as follows. A column waspacked with about 5 ml of Protein A-Sepharose gel and then washed with50 ml of PBS. After the washing, 1 liter of the culture medium of thepAI301-derived transformants described above was applied to the ProteinA-Sepharose column at a flow rate of 0.5 ml/min. Then, the column waswashed with 50 ml of PBS. Thereafter, 20 ml of 0.1 M citrate buffer (pH3.0) were applied to the column to thereby elute the protein adsorbed onProtein A-Sepharose and, simultaneously, fractionate the eluate into1-ml fractions. To each of the fractions, 0.15 ml of 2M Tris-HCl (pH9.0) was added for pH adjustment. The protein concentration of eachfraction was measured using a protein concentration measurement kit(Bio-rad) and those fractions having high protein concentration wererecovered. The resultant protein solution was placed in a dialysis tubeand dialyzed against PBS. Thus, a purified hIL-5R α-Fc (proteinconcentration: 1.8 mg/ml; 5.5 ml) was obtained.

(10) Expression of shIL-5R α or hIL-5R α-Fc in Insect Cells

The expression of shIL-5R α and hIL-5R α-Fc was performed by theprocedures described below according to the manual attached toBaculoGold Starter Kit (Pharmingen).

The recovery of shIL-5R α and hIL-5R α-Fc from culture mediums wasperformed using Concanavalin A-Sepharose andDiethylaminoethyl(DEAE)-Sepharose, or Protein A-Sepharose (allmanufactured by Pharmacia Biotech), respectively.

shIL-5R α was obtained as follows. Briefly, 6×10⁶ Sf9 cells weresuspended in 45 ml of Grace's Insect Medium (Gibco) containing 10% FCSin a 225 cm2 flask (Greiner) and cultured at 27° C. for 3–4 days. Afterthe culture supernatant was removed, 30 ml of a fresh Grace's InsectMedium containing 10% FCS and 1 ml of a solution in which therecombinant virus derived from the transfer vector pAI292 obtained in1(8) of Example 1 were contained at a concentration of approx. 1×10⁷PFU/ml were added. The cells were cultured at 27° C. for one additionalday. Then, after the removal of the culture supernatant, 45 ml of afresh Sf900-II medium were added and the cells were cultured for 2–3days. After the completion of the cultivation, the culture supernatantwas recovered and centrifuged at 1,500×g for 10 minutes, to therebyobtain a supernatant. To the resultant culture medium, sodium chloridewas added to give a final concentration of 0.5 M. Then, 1/50 volume of 1M Tris-HCl (pH 7.4) was added and the pH of the resultant mixture wasadjusted to 7.4 with 1 N sodium hydroxide solution.

A column was packed with about 10 ml of Concanavalin A-Sepharose gel andwashed with 50 ml of a buffer containing 20 mM Tris-HCl (pH 7.4) and 0.5mM sodium chloride at a flow rate of 0.5 ml/min. After the washing, 500ml of the shIL-5R α containing culture medium prepared as describedabove were applied to the Concanavalin A-Sepharose column at a flow rateof 0.5 ml/min. Then, the column was washed with 80 ml of a buffercontaining 20 mM Tris-HCl (pH 7.4) and 0.5 mM sodium chloride at a flowrate of 0.5 ml/min. Thereafter, 60 ml of a buffer containing 1 Mα-methylmannoside, 20 mM Tris-HCl (pH 7.4) and 0.5 M sodium chloridewere applied to the column to thereby elute the protein adsorbed onConcanavalin A-Sepharose and, simultaneously, fractionate the eluateinto 2-ml fractions. The protein concentration of each fraction wasmeasured using a protein concentration measurement kit (Bio-rad). Thosefractions with high protein-concentration were recovered in a totalamount of 44 ml and dialyzed against 20 mM Tris-HCl (pH 7.4). Further,similar operations were performed on 900 ml of the shIL-5R α containingculture medium prepared as described above so as to recover thosefractions with high protein-concentration in a total amount of 40 ml,which were dialyzed against 20 mM Tris-HCl (pH 7.4).

After the dialysis, the two protein solutions were combined and appliedto a column packed with 10 ml of Diethylaminoethyl(DEAE)-Sepharose gelto have the protein adsorbed. The elution of shIL-5R α from the columnwas performed by linearly changing the sodium chloride concentrationfrom 0 to 0.5 M. Thus, those fractions with high concentration ofshIL-5R α were recovered in a total amount of 4 ml. This proteinsolution was placed in a dialysis tube and dialyzed against PBS. Thus, apurified shIL-5R α (protein concentration: 400 μg/ml; 4.5 ml) wasobtained.

In a separate step, hIL-5R α-Fc was obtained as follows. Briefly, 6×10⁶Sf9 cells were suspended in 45 ml of Grace's Insect Medium (Gibco)containing 10% FCS in a 225 cm2 flask (Greiner) and cultured at 27° C.for 3–4 days. After the culture supernatant was removed, 30 ml of afresh Grace's Insect Medium containing 10% FCS and 1 ml of a solution inwhich the recombinant virus derived from the transfer vector pAI297obtained in 1(8) of Example 1 were contained at a concentration ofapprox. 1×10⁷ PFU/ml were added. The cells were cultured further at 27°C. for one additional day. Then, after the removal of the culturesupernatant, 45 ml of a fresh Sf900-II medium were added and the cellswere cultured for 2–3 days. After the completion of the cultivation, theculture supernatant was recovered and centrifuged at 1,500×g for 10minutes, to thereby obtain a supernatant.

A column was packed with about 5 ml of Protein A-Sepharose gel andwashed with 50 ml of PBS. After the washing, 450 ml of the shIL-5R α-Fccontaining culture medium as described above were applied to the ProteinA-Sepharose column at a flow rate of 0.5 ml/min. Then, the column waswashed with 50 ml of PBS. Thereafter, 20 ml of 0.1 M citrate buffer (pH3.0) were applied to the column to thereby elute the protein adsorbed onProtein A-Sepharose and, simultaneously, fractionate the eluate into1-ml fractions. To each of the fractions, 0.15 ml of 2 M Tris-HCl (pH9.0) was added for pH adjustment. The protein concentration of eachfraction was measured using a protein concentration measurement kit(Bio-rad) and those fractions with high protein concentration wererecovered. The thus obtained protein solution was concentrated by afactor of about 3 using Centricon-30 (Amicon), placed in a dialysis tubeand dialyzed against PBS. Thus, a purified shIL-5R α-Fc (proteinconcentration: 0.4 mg/ml; 1.8 ml) was obtained.

(11) Expression of an shIL-5R α Partial Fragment in E. coli

The expression of an shIL-5R α partial fragment in E. coli was performedby inserting a DNA fragment containing a cDNA coding for an shIL-5R αpartial fragment into E. coli expression vector pMKex1 to be describedbelow so as to construct pAI263 and transform E. coli with pAI263.

Briefly, 3 μg of plasmid pGHA2 (Kokai No. Sho 60-221091) were added to30 μl of a buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM magnesiumchloride, 100 mM sodium chloride and 1 mM DTT, to which 10 units ofEcoRI were added and reacted at 37° C. for 4 hours. DNA fragments wererecovered from the reaction mixture by ethanol precipitation. To theseDNA fragments, 30 μl of a buffer containing 10 mM Tris-HCl (pH 7.5), 10mM magnesium chloride, 50 mM sodium chloride and 1 mM DTT, and 10 unitsof ClaI were added and reacted at 37° C. for 4 hours. The reactionmixture was subjected to agarose gel electrophoresis, and about 0.3 μgof the EcoRI-ClaI fragment from pGHA2 containing the promoter region wasrecovered.

Three μg of plasmid pTerm2 (Kokai No. 227075/90) were added to 30 μl ofa buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride,100 mM sodium chloride and 1 mM DTT, to which 10 units of EcoRI wereadded and reacted at 37° C. for 4 hours. DNA fragments were recoveredfrom the reaction mixture by ethanol precipitation. To these DNAfragments, 30 μl of a buffer containing 10 mM Tris-HCl (pH 8.4), 10 mMmagnesium chloride, 100 mM sodium chloride and 1 mM DTT and 10 units ofNsiI were added and reacted at 37° C. for 4 hours. The reaction mixturewas subjected to agarose gel electrophoresis, and about 0.8 μg of theEcoRI-NsiI fragment from pTerm2 was recovered.

Fifty ng of the EcoRI/ClaI fragment from pGHA2, 100 ng of the EcoRI/NsiIfragment from pTerm2 and 100 ng of a synthetic DNA shown in SEQ ID NO:15 were dissolved in 20 μl of T4DNA ligase solution, to which 200 unitsof T4DNA ligase were added. Then, ligation was performed at 12° C. for16 hours. Using the thus prepared recombinant plasmid DNA, E. colistrain JM109 was transformed to thereby obtain plasmid pMKex1 shown inFIG. 12.

In a separate step, 3 μg of pAI234 obtained in FIG. 3 were added to 30μl of a buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM magnesiumchloride, 100 mM sodium chloride and 1 mM DTT, to which 10 units of PstIwere added and reacted at 37° C. for 4 hours. DNA fragments wererecovered from the reaction mixture by ethanol precipitation anddissolved in 20 μl of T4DNA polymerase I buffer [a buffer containing 33mM Tris-HCl (pH 8.0), 66 mM potassium acetate, 10 mM magnesium acetate,0.5 mM DTT and 0.01% BSA]. To the resultant mixture, 5 units of T4DNApolymerase I (Takara Shuzo) were added and reacted at 12° C. for 15minutes, whereby the 5′ cohesive ends generated by the PstI digestionwere changed to blunt ends. The reaction mixture was subjected tophenol-chloroform extraction followed by ethanol precipitation. To theprecipitate, 30 μl of a buffer containing 20 mM Tris-HCl (pH 8.5), 10 mMmagnesium chloride, 100 mM potassium chloride and 1 mM DTT and 10 unitsof Bam-HI were added and reacted at 37° C. for 4 hours. The reactionmixture was subjected to agarose gel electrophoresis, and about 0.3 μgof an approx. 0.7 kb DNA fragment containing a cDNA coding for anshIL-5R α fragment was recovered.

Three μg of the expression vector for E. coli, pMKex1 obtained in FIG.12 were dissolved in 30 μl of a buffer containing 20 mM Tris-HCl (pH8.5), 10 mM magnesium chloride, 100 mM potassium chloride and 1 mM DTT,to which 10 units of BamHI were added and reacted at 37° C. for 4 hours.DNA fragments were recovered from the reaction mixture by ethanolprecipitation and dissolved in 30 μl of a buffer containing 50 mMTris-HCl (pH 7.5), 10 mM magnesium chloride, 100 mM sodium chloride and1 mM DTT, to which 10 units of EcoRV were added and reacted at 37° C.for 4 hours. About 1.5 μg of DNA fragments were recovered from thereaction mixture by ethanol precipitation.

Fifty ng of the thus obtained cDNA coding for an shIL-5R α fragment and100 ng of the thus obtained RcoRV/BamHI fragment from pMKex1 weredissolved in 20 μl of T4DNA ligase buffer, to which 200 units of T4DNAligase were added. Then, ligation was performed at 12° C. for 16 hours.Using the thus prepared recombinant plasmid DNA, E. coli strain JM109was transformed to thereby obtain plasmid pAI263 shown in FIG. 13.

The above plasmid pAI263 was transfected into E. coli (MolecularCloning, A Laboratory Manual, 2nd Edition published by Cold SpringHarbor Laboratory Press, 1989), which was cultured in 400 ml of LBmedium containing 200 μg/ml of ampicillin at 37° C. for 4 hours. Then,0.5 mM IPTG was added and the cells were cultured at 37° C. for another2 hours. Four hundred ml of the culture medium were centrifuged at3,000×g for 15 minutes. The precipitate containing the cells of E. coliwas suspended in 100 ml of buffer I [10 mM Tris-HCl (pH 8.0), 1 mM EDTA,150 mM sodium chloride]. After recentrifugation, the precipitate wassuspended in 7 ml of buffer I and sonicated to disrupt cells. Theresultant suspension was centrifuged at 10,000×g for 30 minutes, and theprecipitate was dissolved in 500 μl of SDS-polyacrylamide gelelectrophoresis sample buffer [6 mM Tris-HCl (pH 6.8), 2% SDS, 10%glycerol, 5% 2-mercaptoethanol] and subjected to polyacrylamide gelelectrophoresis. Thus, a purified shIL-5R α fragment having a molecularweight of about 27 kD was obtained.

(12) Preparation of a Cell Membrane Fraction from Human IL-5R αExpressing Cells

The preparation of a membrane component from the hIL-5R α genetransfected CTLL-2 cells [J. Exp. Med., 177, 1523 (1993)] or controlCTLL-2 cells [ATCC TIB 214] was performed as described below.

Briefly, the cells were centrifuged (1,200 rpm, 5 min.), washed with PBStwice, and then suspended in cell disruption buffer [20 mM HEPES (pH7.4), 1 mM EDTA, 0.5 mM PMSF, 250 mM sucrose] and disrupted with ahomogenizer. After the disruption, the cells were centrifuged at 5,500rpm for 15 minutes to remove the precipitate. The cells were furthercentrifuged at 35,000 rpm to recover cell membrane fractions as aprecipitate.

2. Immunization of Animals and Preparation of Antibody-Producing Cells

Fifty μg of each of the antigens obtained in subsections (9), (10), (11)or (12) of 10 section 1 of Example 1 were administered independently to5-week old female BALB/c mice or female SD rats together with 2 mg ofaluminum gel and 1×10⁹ cells of pertussis vaccine (Chiba PrefecturalSerum Research Institute). 2 weeks after the administration, 50 μg ofthe protein were administered once a week in total of 4 times. Bloodsamples were collected from the venous plexus of eyegrounds or the tailvein, and antibody titer of the serum thereof was examined by the enzymeimmunoassay described below under 3. Spleens were removed 3 days afterthe final immunization from those mice or rats which exhibited asufficient antibody titer. In this immunization experiment, the cellmembrane fraction obtained in subsection (12) of section 1 of Example 1was used as an antigen to immunize 13 mice and 5 rats. However, noremarkable rise in antibody titer was observed in those animals. Also,no satisfactory rise in antibody titer was observed in the 5 ratsimmunized with the shIL-5R α obtained in subsection (9) of section 1 ofExample 1 or the 10 rats immunized with the shIL-5R α obtained insubsection (10) of section 1 of Example 1.

The spleen was cut into pieces in MEM medium (Nissui Pharmaceuticals),loosened with tweezers and centrifuged (1,200 rpm, 5 min.). Then, thesupernatant was discarded and the remainder was treated withTris-ammonium chloride buffer (pH 7.65) for 1–2 minutes to removeerythrocytes and washed with MEM medium 3 times. The resultantsplenocytes were used for cell fusion.

3. Enzyme Immunoassay

The measurement of antisera or culture supernatants of hybridoma cellsderived from mice or rats immunized with the shIL-5R α obtained insubsections (9) or (10) of section 1 of Example 1 was performedaccording to the two methods described below using, as an antigen, thehIL-5R α-Fc obtained from a culture supernatant of insect cells asdescribed in subsection (10) of section 1 Example 1.

-   -   (A) To a 96-well EIA plate (Greiner), hIL-5R α-Fc diluted to 1        μg/ml with PBS and a control antigen, anti-GD3 chimeric antibody        KM871 having a common human Ig constant region, were dispensed        separately in an amount of 50 μl/well and left at 4° C.        overnight to have the proteins adsorbed. After washing, PBS        containing 1% bovine serum albumin (BSA) (hereinafter, referred        to as 1% BSA-PBS) was added to the plate (100 μl/well) and        reacted at room temperature for 1 hour to thereby block the        remaining active groups. After discarding 1% BSA-PBS, an        immunized mouse or rat-derived antiserum and culture supernatant        of a hybridoma were dispensed into the wells (50 μl/well) and        reacted for 2 hours. After washing with Tween-PBS,        peroxidase-labeled rabbit anti-mouse immunoglobulin or anti-rat        immunoglobulin (DAKO) was added to the plate (50 μl/well),        reacted for 1 hour and washed with Tween-PBS. Thereafter, the        resultant mixture was allowed to form a color by using ABTS        substrate solution [a solution obtained by dissolving 550 mg of        2,2′ azinobis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium        salt in 1 L of 0.1 M citrate buffer (pH 4.2) and adding 1 μl/ml        of hydrogen peroxide immediately before use] to measure the        absorbance at OD415 nm (NJ2001; Japan Intermed).    -   (B) Further, for the purpose of selecting a monoclonal antibody        having neutralizing activity against IL-5 with a higher        probability, screening was performed for an activity to inhibit        binding to an IL-5 receptor by the following procedures using a        biotin-labeled human IL-5 and the shIL-5R α-Fc obtained from the        insect cell culture supernatant in subsection (10) of section 1        of Example 1. The human IL-5 used for biotin labeling was        prepared according to the method described in Journal of        Immunological Method, 125, 233 (1989).

The biotin labeling of the human IL-5 was performed according to theprotocol attached to a biotin-labeling reagent (Biotin-LC-Hydrazide)(Pierce) by the following procedures. First, 1.6 mg/ml of human IL-5dissolved in PBS was applied to a PD10 column (Pharmacia) equilibratedwith a labeling buffer (100 mM sodium acetate, 0.02% NaN3, pH 5.5) forsalt exchange and 1 ml of a fraction having high protein concentrationwas recovered. To 0.5 ml of this human IL-5 solution, 1 ml of a labelingbuffer containing 30 mM metaperiodic acid was added and reacted at roomtemperature for 30 minutes while shielding the light. After thecompletion of the reaction, the reaction mixture was applied to a PD10column equilibrated with a labeling buffer to remove the unreactedmetaperiodic acid. Thus, 1.5 ml of a fraction having high proteinconcentration was recovered. To this fraction, 20 μl of a labelingbuffer containing 5 mM biotin-labeling reagent as described above wereadded and reacted at room temperature for 1 hour. After the completionof the reaction, 50 μl of reaction termination buffer (0.1 M Tris, pH7.5) were added, and then the reaction mixture was applied to a PD10column equilibrated with 0.05% NaN3-containing PBS to exchange saltsand, simultaneously, remove unreacted reagents. The thus obtainedbiotin-labeled human IL-5 was stored at 4° C.

The shIL-5R α-Fc obtained from the insect cell culture supernatant insubsection (10) of section 1 of Example 1 was diluted to a concentrationof 5 μg/ml with PBS, dispensed into a 96-well EIA plate (Greiner) (50μl/well) and left at 4° C. overnight to have the protein adsorbed. Afterwashing with PBS, PBS containing 1% bovine serum albumin (BSA) (1%BSA-PBS) was added to the plate (100 μl/well) and reacted at roomtemperature for 1 hour to block the remaining active groups. Then, theplate was washed with Tween-PBS. Thereafter, an antiserum derived fromimmunized mouse or rat and the culture supernatant of the hybridoma, andthe biotin-labeled human IL-5 described above were each added to theplate in an amount of 50 μl/well and reacted at 4° C. overnight. On thenext day, the plate was washed with Tween-PBS, and then 50 μl/well ofperoxidase-labeled avidin (Nippon Reizo) diluted 4000 folds with 1%BSA-PBS were added and reacted at room temperature for 1 hour. Afterwashing with Tween-PBS, 50 μl/well of ABTS substrate solution were addedto allow color development and the absorbance at OD415 was measured.

With respect to the measurement of antisera and culture supernatants ofhybridomas derived from those mice or rats immunized with the hIL-5R αfragment obtained in subsection (11) of section 1 of Example 1, thehIL-5R α fragment produced by E. coli in subsection (11) of section 1 ofExample 1 was used as an antigen. In a manner similar to that describedabove, the shIL-5R α produced by E. coli and an E. coli cell protein(control antigen) were adsorbed on plates separately. Using thusprepared plates, the reactivity of culture supernatants of hybridomasand antisera of immunized mice or rats was examined.

Further, with respect to the measurement of antisera and culturesupernatants of hybridomas derived from those mice or rats immunizedwith the cell membrane fraction from hIL-5R α expressing cells obtainedin subsection (12) of section 1 of Example 1, the cell membrane fractionobtained in subsection (12) of section 1 of Example 1 was used as anantigen. In a manner similar to that described above, the cell membranefraction from IL-5R α-expressing cells and a cell membrane fraction fromcontrol cells were adsorbed on plates separately. Using thus preparedplates, the reactivity of culture supernatants of hybridomas andantisera of immunized mice or rats was examined.

4. Preparation of Mouse Myeloma Cells

An 8-azaguanine resistant mouse myeloma cell line, P3-U1, was culturedin a normal medium and not less than 2×10⁷ cells were secured andsubmitted for cell fusion as a parent line.

5. Preparation of Hybridomas

The mouse or rat splenocytes obtained in section 2 of Example 1 and themyeloma cells obtained in section 4 of Example 1 were mixed at a ratioof 10:1, and the mixture was centrifuged (1,200 rpm, 5 min.). Then, thesupernatant was discarded and the precipitated cells were loosenedsufficiently. To the resultant cells, a mixed solution composed of 2 gof polyethylene glycol-1000 (PEG-1000), 2 ml of MEM medium and 0.7 ml ofDMSO was added in an amount of 0.2 to 1 ml per 10⁸ mouse splenocytes,followed by the addition of 1 to 2 ml portions of MEM medium at 1 to 2minute interval at 37° C. Thereafter, MEM medium was added to give atotal volume of 50 ml. After centrifugation (900 rpm, 5 min.), thesupernatant was discarded and cells were loosened gently. Then, cellswere gently suspended in 100 ml of HAT medium by suction and releasewith a pipette.

This cell suspension was dispensed into a 96-well culture plate (100μl/well) and cultured in a 5% CO₂ incubator at 37° C. for 10–14 days.The resultant culture supernatant was examined by the enzyme immunoassaydescribed in section 3 of Example 1, and those wells which showedspecific reaction with the hIL-5R α-Fc prepared from an insect cellculture supernatant or with the shIL-5R α produced by E. coli wereselected. Further, the medium was replaced with HT medium and a normalmedium, and cloning was repeated twice. As a result, hybridoma celllines producing an anti-human IL-5R α monoclonal antibody wereestablished.

As a result of screening about 4000 hybridoma clones obtained from 6mice or 8 rats immunized with the hIL-5R α fragment obtained insubsection (11) of section 1 of Example 1, an anti-human IL-5R αmonoclonal antibody was obtained and designated as KM1074. Itsreactivity with IL-5R α was extremely weak compared to that ofanti-human IL-5R α monoclonal antibodies KM1257 and KM1259 to bedescribed later.

In a separate step, hybridomas were obtained from 12 or 6 animals thatexhibited a high antibody titer and which were selected from 15 or 20mice immunized with the shIL-5R α obtained in subsection (9) of section1 of Example 1 or the shIL-5R α obtained in subsection (10) of section 1of Example 1. As a result of screening more than 10000 hybridoma clones,81 hybridoma clones were established that produced an anti-human IL-5R αmonoclonal antibody and which showed a specific reactivity with hIL-5R αexpressing cells when tested by the method described later in section 1of Example 3. Among these, the monoclonal antibody which exhibited themost strong reactivity in the immunocyte staining method described laterin section 1 of Example 3 later was KM1257. Hybridoma KM1257 wasdeposited at the National Institute of Bioscience and Human-Technology,Agency of Industrial Science and Technology (1-3, Higashi 1-Chome,Tsukuba City, Ibaraki, Japan; hereinafter, the address is the same forthis Institute) on Jun. 13, 1995 under accession number FERM BP-5133. Ofthose 81 clones, only six clones exhibited a strong inhibition activityagainst the biological activity of IL-5 which is described later insection 2 of Example 3. Among these six clones, the monoclonalantibodies which exhibited the strongest inhibition activity were KM1259and KM1486. Hybridoma KM1259 was deposited under accession number FERMBP-5134 on Jun. 13, 1995 and hybridoma KM1486 was deposited underaccession number FERM BP-5651 on Sep. 3, 1996 both at the NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology.

The reactivities of monoclonal antibodies KM1257, KM1259 and KM1486 areshown in FIG. 14. Subclass of each antibody was determined by an enzymeimmunoassay using a subclass typing kit. As a result, the antibodyclasses of KM1257, KM1259 and KM1486 were all IgG1.

6. Purification of Monoclonal Antibodies

The hybridoma cell line obtained in 5 above was intraperitoneallyadministered to pristane-treated, female nude mice (Balb/c) of 8 weeksof age at a dose of (5–20×10⁶ cells/mouse). The hybridoma caused ascitestumor 10 to 21 days after the administration. From those mice in whichascites accumulated, ascites was collected (1–8 ml/mouse), centrifuged(3,000 rpm, 5 min.) to remove the solids and then purified by thecaprylic acid precipitation method (Antibodies—A Laboratory Manual, ColdSpring Harbor Laboratory, 1988) to obtain purified monoclonal antibody.

Example 2 Preparation of Anti-Human IL-5R α Humanized Antibodies

1. Construction of Tandem Cassette-Type Humanized Antibody ExpressionVector pKANTEX93

A tandem cassette-type humanized antibody expression vector, pKANTEX93,for expressing a humanized antibody of human antibody IgG1, κ type inanimal cells and into which a cDNA coding for a humanized antibody VHand a cDNA coding for a humanized antibody VL were transfected upstreamof a cDNA coding for human antibody Cγ1 and a cDNA coding for humanantibody Cκ, respectively, was constructed as described below based onthe plasmid pSE1UK1SEd1-3 disclosed in Kokai No. 257891/90. Thehumanized antibody expression vector constructed was used for theexpression of human chimeric antibodies and human CDR-grafted antibodiesin animal cells.

(1) Modification of the ApaI and EcoRI Restriction Sites present inRabbit β-Globin Gene Splicing Signal and Poly (A) Signal

The modification of the ApaI and EcoRI restriction sites present inrabbit β-globin gene splicing poly (A) signal of plasmid pSE1UK1SEd1-3was performed as described below in order to enable the construction ofa human chimeric antibody expression vector or a human CDR-graftedantibody (=humanized antibody) expression vector by inserting into ahumanized antibody expression vector the variable region of a humanchimeric antibody or a human CDR-grafted antibody in a cassette using aNotI-ApaI fragment (VH) and an EcoRI-Sp1I fragment (VL).

Briefly, 3 μg of plasmid pBluescript SK(−) (Stratagene) were added to 10μl of a buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM magnesiumchloride and 1 mM DTT, to which 10 units of the restriction enzyme ApaI(Takara Shuzo) were added and reacted at 37° C. for 1 hour. The reactionmixture was ethanol-precipitated, and the 3′ sticky ends generated bythe ApaI digestion were blunted using DNA Blunting Kit (Takara Shuzo)and the resultant DNA fragments were ligated using DNA Ligation Kit(Takara Shuzo). Using the thus obtained recombinant plasmid DNAsolution, E. coli HB101 was transformed to obtain plasmid pBSA shown inFIG. 15.

Further, 3 μg of the thus obtained plasmid pBSA were added to 10 μl of abuffer containing 50 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride, 100mM sodium chloride and 1 mM DTT, to which 10 units of the restrictionenzyme EcoRI (Takara Shuzo) were added and reacted at 37° C. for 1 hour.The reaction mixture was ethanol-precipitated, and the 5′ sticky endsgenerated by the EcoRI digestion were blunted using DNA Blunting Kit(Takara Shuzo) and the resultant DNA fragments were ligated using DNALigation Kit (Takara Shuzo). Using the thus obtained recombinant plasmidDNA solution, E. coli HB101 was transformed to obtain plasmid pBSAEshown in FIG. 16.

Subsequently, 3 μg of the thus obtained plasmid pBSAE were added to 10μl of a buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM magnesiumchloride, 50 mM sodium chloride and 1 mM DTT, to which 10 units of therestriction enzyme HindIII (Takara Shuzo) were added and reacted at 37°C. for 1 hour. The reaction mixture was ethanol-precipitated, and theprecipitate was dissolved in 20 μl of a buffer containing 10 mM Tris-HCl(pH 7.5), 10 mM magnesium chloride and 1 mM DTT. The resultant mixturewas divided into two 10 μl portions. To one portion, 10 units of therestriction enzyme SacII (Toyobo) were added, and to the other portion,10 units of the restriction enzyme KpnI (Takara Shuzo) were added. Then,both mixtures were reacted at 37° C. for 1 hour. Both reaction mixtureswere subjected to agarose gel electrophoresis, and an approx. 2.96 kbHindIII-SacII fragment and an approx. 2.96 kb KpnI-HindIII fragment wererecovered, each in about 0.3 μg.

Subsequently, 3 μg of plasmid pSE1UK1SEd1-3 were added to 10 μl of abuffer containing 10 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride and1 mM DTT, to which 10 units of the restriction enzyme SacII (Toyobo) and10 units of the restriction enzyme KpnI (Takara Shuzo) were added andreacted at 37° C. for 1 hour. The reaction mixture wasethanol-precipitated, and the precipitate was dissolved in 10 μl of abuffer containing 10 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride, 50mM sodium chloride and 1 mM DTT. To the resultant mixture, 10 units ofthe restriction enzyme HindIII (Takara Shuzo) were added and reacted at37° C. for 1 hour. The reaction mixture was subjected to agarose gelelectrophoresis, and an approx. 2.42 kb HindIII-SacII fragment and anapprox. 1.98 kb KpnI-HindIII fragment were recovered, each in about 0.2μg.

Then, 0.1 μg of the HindIII-SacII fragment from plasmid pSE1UK1SEd1-3and 0.1 μg of the HindIII-SacII fragment from pBSAE obtained above weredissolved in sterilized water to give a total volume of 20 μl andligated using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). Using thethus obtained recombinant plasmid DNA solution, E. coli HB101 wastransformed to obtain plasmid pBSH-S shown in FIG. 17. Also, 0.1 μg ofthe KpnI-HindIII fragment from plasmid pSE1UK1SEd1-3 and 0.1 μg of theKpnI-HindIII fragment from pBSAE obtained above were dissolved insterilized water to give a total volume of 20 μl and ligated usingReady-To-Go T4 DNA Ligase (Pharmacia Biotech). Using the thus obtainedrecombinant plasmid DNA solution, E. coli HB101 was transformed toobtain plasmid pBSK-H shown in FIG. 18.

Subsequently, 3 μg each of the thus obtained plasmids pBSH-S and pBSK-Hwere added separately to 10 μl of a buffer containing 10 mM Tris-HCl (pH7.5), 10 mM magnesium chloride and 1 mM DTT, to which 10 units of therestriction enzyme ApaI (Takara Shuzo) were added and reacted at 37° C.for 1 hour. Both reaction mixtures were ethanol-precipitated, and the 3′sticky ends generated by the ApaI digestion were blunted using DNABlunting Kit (Takara Shuzo) and the resultant DNA fragments were ligatedusing DNA Ligation Kit (Takara Shuzo). Using each of the thus obtainedrecombinant plasmid DNA solutions, E. coli HB101 was transformed toobtain plasmid pBSH-SA and pBSK-HA shown in FIG. 19.

Subsequently, 5 μg each of the thus obtained plasmids pBSH-SA andpBSK-HA were added separately to 10 μl of a buffer containing 50 mMTris-HCl (pH 7.5), 10 mM magnesium chloride, 100 mM sodium chloride and1 mM DTT, to which 10 units of the restriction enzyme EcoRI (TakaraShuzo) were added and reacted at 37° C. for 10 minutes so that theplasmid was partially digested. Then, both reaction mixtures wereethanol-precipitated. After the 5′ sticky ends generated by the EcoRIdigestion were blunted using DNA Blunting Kit (Takara Shuzo), bothreaction mixtures were subjected to agarose gel electrophoresis, and anapprox. 5.38 kb fragment and an approx. 4.94 kb fragment were recovered,each in about 0.5 μg. Then, 0.1 μg each of the thus recovered fragmentswere dissolved separately in sterilized water to give a total volume of20 μl and ligated using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech).Using each of the thus obtained recombinant plasmid DNA solutions, E.coli HB101 was transformed to obtain plasmids pBSH-SAE and pBSK-HAEshown in FIG. 20.

Subsequently, 3 μg each of the thus obtained plasmids pBSH-SAE andpBSK-HAE were added separately to 10 μl of a buffer containing 50 mMTris-HCl (pH 7.5), 10 mM magnesium chloride, 100 mM sodium chloride and1 mM DTT, to which 10 units of the restriction enzyme EcoRI (TakaraShuzo) were added and reacted at 37° C. for 1 hour. Both reactionmixtures were ethanol-precipitated and the 5′ sticky ends generated bythe EcoRI digestion were blunted using DNA Blunting Kit (Takara Shuzo)and the resultant DNA fragments were ligated using DNA Ligation Kit(Takara Shuzo). Using each of the thus obtained recombinant plasmid DNAsolutions, E. coli HB101 was transformed to obtain plasmids pBSH-SAEEand pBSK-HAEE shown in FIG. 21. Ten μg each of the thus obtainedplasmids were separately reacted according to the recipe attached toAutoRead Sequencing Kit (Pharmacia Biotech) and then electrophoresedwith A.L.F. DNA Sequencer (Pharmacia Biotech) to thereby determine thebase sequence. As a result, it was confirmed that both the ApaI andEcoRI restriction sites had been eliminated by the above-describedmodification.

(2) Introduction of a SalI Restriction Site into the Downstream Portionconsisting of the Rabbit β-Globin Gene Splicing Signal, Rabbit β-GlobinGene Poly (A) Signal and SV40 Early Gene Poly (A) Signal

In order to ensure that expression promoters for the human antibody Hand L chains in a humanized antibody expression vector could be replacedwith any promoters, a SalI restriction site was transfected into thedownstream portion consisting of the rabbit β-globin gene splicingsignal, rabbit β-globin gene poly (A) signal and SV40 early gene poly(A) signal of plasmid pSE1UK1SEd1-3 as described below.

Briefly, 3 μg of the plasmid pBSK-HAEE obtained in subsection (1) ofsection 1 of Example 2 were added to 10 μl of a buffer containing 10 mMTris-HCl (pH 7.5), 10 mM magnesium chloride and 1 mM DTT, to which 10units of the restriction enzyme NaeI (Takara Shuzo) were added andreacted at 37° C. for 1 hour. The reaction mixture wasethanol-precipitated and the precipitate was dissolved in 20 μl of abuffer containing 50 mM Tris-HCl (pH 9.0) and 1 mM magnesium chloride,to which 1 unit of alkaline phosphatase (E. coli C75, Takara Shuzo) wasadded and reacted at 37° C. for 1 hour to dephosphorylate 5′ ends. Then,the reaction mixture was subjected to phenol-chloroform extraction,followed by ethanol precipitation. The precipitate was dissolved in 20μl of a buffer containing 10 mM Tris-HCl (pH 8.0) and 1 mMethylenediamine-tetraacetic acid disodium (hereinafter referred to as“TE buffer”). One μl of the mixture and 0.1 μg of a phosphorylated SalIlinker (Takara Shuzo) were added to sterilized water to give a totalvolume of 20 μl, and ligated using Ready-To-Go T4 DNA Ligase (PharmaciaBiotech). Using the thus obtained recombinant plasmid DNA solution, E.coli HB101 was transformed to obtain plasmids pBSK-HAEESal shown in FIG.22. Ten μg each of the thus obtained plasmid were reacted according tothe recipe attached to AutoRead Sequencing Kit (Pharmacia Biotech) andthen electrophoresed with A.L.F. DNA Sequencer (Pharmacia Biotech) tothereby determine the base sequence. As a result, it was confirmed thatone SalI restriction site had been transfected into the downstreamportion consisting of the rabbit β-globin gene splicing signal, rabbitβ-globin gene poly (A) signal and SV40 early gene poly (A) signal.

(3) Modification of the ApaI Restriction Site present in the Poly (A)Signal of Herpes Simplex Virus Thymidine Kinase (hereinafter referred toas “HSVtk”) Gene

The modification of the ApaI restriction site present in the poly (A)signal of HSVtk gene located downstream of Tn5 kanamycinphosphotransferase gene in plasmid pSE1UK1SEd1-3 was performed asdescribed below.

Briefly, 3 μg of the plasmid pBSA obtained in subsection (1) of section1 of Example 2 were added to 10 μl of a buffer containing 10 mM Tris-HCl(pH 7.5), 10 mM magnesium chloride and 1 mM DTT, to which 10 units ofthe restriction enzyme SacII (Toyobo) were added and reacted at 37° C.for 1 hour. The reaction mixture was ethanol-precipitated and theprecipitate was dissolved in 10 μl of a buffer containing 50 mM Tris-HCl(pH 7.5), 100 mM sodium chloride, 10 mM magnesium chloride and 1 mM DTT,to which 10 units of the restriction enzyme XhoI (Takara Shuzo) wereadded and reacted at 37° C. for 1 hour. The reaction mixture wassubjected to agarose gel electrophoresis and about 1 μg of an approx.2.96 kb SacII-XhoI fragment was recovered.

Subsequently, 5 μg of plasmid pSE1UK1SEd1-3 were added to 10 μl of abuffer containing 10 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride and1 mM DTT, to which 10 units of the restriction enzyme SacII (Toyobo)were added and reacted at 37° C. for 1 hour. The reaction mixture wasethanol-precipitated and the precipitate was dissolved in 10 μl of abuffer containing 50 mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 10 mMmagnesium chloride and 1 mM DTT, to which 10 units of the restrictionenzyme XhoI (Takara Shuzo) were added and reacted at 37° C. for 1 hour.The reaction mixture was subjected to agarose gel electrophoresis andabout 1 μg of an approx. 4.25 kb SacII-XhoI fragment was recovered.

Subsequently, 0.1 μg of the SacII-XhoI fragment from pBSA and theSacII-XhoI fragment from plasmid pSE1UK1SEd1-3 as obtained above wereadded to sterilized water to give a total volume of 20 μl, and thenligated using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). Using thethus obtained recombinant plasmid DNA solution, E. coli HB101 wastransformed to obtain plasmid pBSX-S shown in FIG. 23.

Subsequently, 3 μg of the thus obtained plasmid pBSX-X were added to 10μl of a buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM magnesiumchloride and 1 mM DTT, to which 10 units of the restriction enzyme ApaI(Takara Shuzo) were added and reacted at 37° C. for 1 hour. The reactionmixture was ethanol-precipitated, and the 3′ sticky ends generated bythe ApaI digestion were blunted using DNA Blunting Kit (Takara Shuzo)and the resultant DNA fragments were ligated using DNA Ligation Kit(Takara Shuzo). Using the thus obtained recombinant plasmid DNAsolution, E. coli HB101 was transformed to obtain plasmid pBSX-SA shownin FIG. 24. Ten μg of the thus obtained plasmid were reacted accordingto the recipe attached to AutoRead Sequencing Kit (Pharmacia Biotech)and then electrophoresed with A.L.F. DNA Sequencer (Pharmacia Biotech)to thereby determine the base sequence. As a result, it was confirmedthat the ApaI restriction site of the HSVtk gene poly (A) signal hadbeen eliminated.

(4) Construction of a Humanized Antibody L Chain Expression Unit

Plasmid mMohCκ having a humanized antibody L chain expression unit inwhich a cDNA coding for the constant region of human κ-type L-chain (Cκ)was located downstream of the promoter/enhancer of the terminal repeatedsequence of Moloney mouse leukemia virus and into which a cDNA codingfor VL of a human chimeric antibody or human CDR-grafted antibody couldbe inserted in a cassette was constructed as described below.

Briefly, 3 μg of plasmid pBluescript SK(−) (Stratagene) were added to 10μl of a buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM magnesiumchloride and 1 mM DTT, to which 10 units of the restriction enzyme SacI(Takara Shuzo) were added and reacted at 37° C. for 1 hour. The reactionmixture was ethanol-precipitated, and the precipitate was added to 10 μlof a buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM sodium chloride,10 mM magnesium chloride and 1 mM DTT, to which 10 units of therestriction enzyme ClaI (Takara Shuzo) were added and reacted at 37° C.for 1 hour. The reaction mixture was ethanol-precipitated, and thesticky ends generated by the SacI and ClaI digestions were blunted usingDNA Blunting Kit (Takara Shuzo). Then, the reaction mixture wassubjected to agarose gel electrophoresis to thereby recover about 1 μgof an approx. 2.96 kb DNA fragment. Then, 0.1 μg of the recovered DNAfragment was added to sterilized water to give a total volume of 20 μland ligated using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). Usingthe thus obtained recombinant plasmid DNA solution, E. coli HB101 wastransformed to obtain plasmid pBSSC shown in FIG. 25.

Subsequently, 3 μg of the thus obtained plasmid pBSSC were added to 10μl of a buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM magnesiumchloride and 1 mM DTT, to which 10 units of the restriction enzyme KpnI(Takara Shuzo) were added and reacted at 37° C. for 1 hour. The reactionmixture was ethanol-precipitated, and the precipitate was dissolved in10 μl of a buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM sodiumchloride, 10 mM magnesium chloride and 1 mM DTT, to which 10 units ofthe restriction enzyme XhoI (Takara Shuzo) were added and reacted at 37°C. for 1 hour. Then, the reaction mixture was subjected to agarose gelelectrophoresis to thereby recover about 1 μg of an approx. 2.96 kbKpnI-XhoI fragment.

Subsequently, 5 μg of the plasmid pAGE147 disclosed in Kokai No.205694/94 were added to 10 μl of a buffer containing 10 mM Tris-HCl (pH7.5), 10 mM magnesium chloride and 1 mM DTT, to which 10 units of therestriction enzyme KpnI (Takara Shuzo) were added and reacted at 37° C.for 1 hour. The reaction mixture was ethanol-precipitated, and theprecipitate was dissolved in 10 μl of a buffer containing 50 mM Tris-HCl(pH 7.5), 100 mM sodium chloride, 10 mM magnesium chloride and 1 mM DTT,to which 10 units of the restriction enzyme XhoI (Takara Shuzo) wereadded and reacted at 37° C. for 1 hour. Then, the reaction mixture wassubjected to agarose gel electrophoresis to thereby recover about 0.3 μgof an approx. 0.66 kb KpnI-XhoI fragment containing thepromoter/enhancer of the terminal repeated sequence of Moloney mouseleukemia virus.

Subsequently, 0.1 μg of the KpnI-XhoI fragment from pBSSC and 0.1 μg ofthe KpnI-XhoI fragment from pAGE147 as obtained above were dissolved insterilized water to give a total volume of 20 μl and ligated usingReady-To-Go T4 DNA Ligase (Pharmacia Biotech). Using the thus obtainedrecombinant plasmid DNA solution, E. coli HB101 was transformed toobtain plasmid pBSMo shown in FIG. 26.

Subsequently, 3 μg of the plasmid pBSMo obtained above were added to 10μl of a buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM magnesiumchloride and 1 mM DTT, to which 10 units of the restriction enzyme KpnI(Takara Shuzo) were added further and reacted at 37° C. for 1 hour. Thereaction mixture was ethanol-precipitated, and the precipitate wasdissolved in 10 μl of a buffer containing 10 mM Tris-HCl (pH 7.5), 50 mMsodium chloride, 10 mM magnesium chloride and 1 mM DTT, to which 10units of the restriction enzyme HindIII (Takara Shuzo) were addedfurther and reacted at 37° C. for 1 hour. Then, the reaction mixture wassubjected to agarose gel electrophoresis to thereby recover about 1 μgof an approx. 3.62 kb KpnI-HindIII fragment.

Subsequently, two synthetic DNAs having the base sequences shown in SEQID NOS: 16 and 17, respectively, were synthesized using an automatic DNAsynthesizer (380A, Applied Biosystems). Then, 0.3 μg each of thesynthetic DNAs obtained were added to 15 μl of sterilized water andheated at 65° C. for 5 minutes. The reaction mixture was left at roomtemperature for 30 minutes. To this mixture, 2 μl of a 10×buffer [500 mMTris-HCl (pH 7.6), 100 mM magnesium chloride, 50 mM DTT] and 2 μl of 10mM ATP were added. Further, 10 units of T4 polynucleotide kinase (TakaraShuzo) were added and reacted at 37° C. for 30 minutes to phosphorylatethe 5′ ends. Then, 0.1 μg of the KpnI-HindIII fragment (3.66 kb) fromplasmid pBSMo as obtained above and 0.05 μg of the phosphorylatedsynthetic DNAs were added to sterilized water to give a total volume of20 μl and ligated using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech).Using the thus obtained recombinant plasmid DNA solution, E. coli HB101was transformed to obtain plasmid pBSMoS shown in FIG. 27. Ten μg of thethus obtained plasmid were reacted according to the recipe attached toAutoRead Sequencing Kit (Pharmacia Biotech) and then electrophoresedwith A.L.F. DNA Sequencer (Pharmacia Biotech) to thereby determine thebase sequence. As a result, it was confirmed that the synthetic DNAs ofinterest had been transfected.

Subsequently, 3 μg of the plasmid pChilgLA1 disclosed in Kokai No.304989/93 were dissolved in 10 μl of a buffer containing 50 mM Tris-HCl(pH 7.5), 100 mM sodium chloride, 10 mM magnesium chloride and 1 mM DTT,to which 10 units each of the restriction enzymes EcoRI (Takara Shuzo)and RcoRV (Takara Shuzo) were added and reacted at 37° C. for 1 hour.The reaction mixture was subjected to agarose gel electrophoresis tothereby recover about 1 μg of an approx. 9.70 kb EcoRI-EcoRV fragment.Subsequently, two synthetic DNAs having the base sequences shown in SEQID NOS: 18 and 19, respectively, were synthesized using an automatic DNAsynthesizer (380A, Applied Biosystems). Then, 0.3 μg each of theobtained synthetic DNAs were added to 15 μl of sterilized water andheated at 65° C. for 5 minutes. The reaction mixture was left at roomtemperature for 30 minutes. To this solution, 2 μl of a 10×buffer [500mM Tris-HCl (pH 7.6), 100 mM magnesium chloride, 50 mM DTT] and 2 μl of10 mM ATP were added. Further, 10 units of T4 polynucleotide kinase(Takara Shuzo) were added and reacted at 37° C. for 30 minutes tophosphorylate the 5′ ends. Then, 0.1 μg of the EcoRI-EcoRV fragment(9.70 kb) from plasmid pChiIgLA1 as obtained above and 0.05 μg of thephosphorylated synthetic DNAs were added to sterilized water to give atotal volume of 20 μl and ligated using Ready-To-Go T4 DNA Ligase(Pharmacia Biotech). Using the thus obtained recombinant plasmid DNAsolution, E. coli HB101 was transformed to obtain plasmid pChilgLA1Sshown in FIG. 28.

Subsequently, 3 μg of the plasmid pBSMoS as obtained above weredissolved in 10 μl of a buffer containing 20 mM Tris-HCl (pH 8.5), 100mM potassium chloride, 10 mM magnesium chloride and 1 mM DTT, to which10 units of the restriction enzyme HpaI (Takara Shuzo) were added andreacted at 37° C. for 1 hour. The reaction mixture wasethanol-precipitated, and the precipitate was dissolved in 10 μl of abuffer containing 50 mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 10 mMmagnesium chloride and 1 mM DTT, to which 10 units of the restrictionenzyme EcoRI (Takara Shuzo) were added and reacted at 37° C. for 1 hour.Then, the reaction mixture was subjected to agarose gel electrophoresisto recover about 1 μg of an approx. 3.66 kb HpaI-EcoRI fragment.

Subsequently, 10 μg of the plasmid pChiIgLA1S as obtained above weredissolved in 10 μl of a buffer containing 20 mM Tris-HCl (pH 7.9), 50 mMpotassium acetate, 10 mM magnesium acetate, 1 mM DTT and 100 μ/ml BSA,to which 10 units of the restriction enzyme NlaIV (New England Biolabs)were added and reacted at 37° C. for 1 hour. The reaction mixture wasethanol-precipitated, and the precipitate was dissolved in 10 μl of abuffer containing 50 mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 10 mMmagnesium chloride and 1 mM DTT, to which 10 units of the restrictionenzyme EcoRI (Takara Shuzo) were added and reacted at 37° C. for 1 hour.Then, the reaction mixture was subjected to agarose gel electrophoresisto recover about 0.3 μg of an approx. 0.41 kb NlaIV-EcoRI fragment.

Subsequently, 0.1 μg each of the HpaI-EcoRI fragment from pBSMoS and theNlaIV-EcoRI fragment from pChiIgLA1S as obtained above were added tosterilized water to give a total volume of 20 μl and ligated usingReady-To-Go T4 DNA Ligase (Pharmacia Biotech). Using the thus obtainedrecombinant plasmid DNA solution, E. coli HB101 was transformed toobtain plasmid pMohCκ shown in FIG. 29.

(5) Construction of a Humanized Antibody H Chain Expression Unit

Plasmid mMohCγ1 having a humanized antibody H chain expression unit inwhich a cDNA coding for the constant region of human IgG1 type H-chain(Cγ1) was located downstream of the promoter/enhancer of the terminalrepeated sequence of Moloney mouse leukemia virus and into which a cDNAcoding for VH of a human chimeric antibody or human CDR-grafted antibodycould be inserted in a cassette was constructed as described below.

Briefly, 3 μg of the plasmid pBSMo obtained in subsection (4) of section1 of Example 2 were added to 10 μl of a buffer containing 50 mM Tris-HCl(pH 7.5), 100 mM sodium chloride, 10 mM magnesium chloride and 1 mM DTT,to which 10 units of the restriction enzyme XhoI (Takara Shuzo) wereadded and reacted at 37° C. for 1 hour. The reaction mixture wasethanol-precipitated, and the precipitate was dissolved in 10 μl of abuffer containing 30 mM sodium acetate (pH 5.0), 100 mM sodium chloride,1 mM zinc acetate and 10% glycerol, to which 10 units of mung beannuclease (Takara Shuzo) were added and reacted at 37° C. for 10 minutes.The reaction mixture was subjected to phenol-chloroform extraction,followed by ethanol precipitation. Then, the sticky ends were bluntedusing DNA Blunting Kit (Takara Shuzo) and the resultant DNA fragmentswere ligated using DNA Ligation Kit (Takara Shuzo). Using the thusobtained recombinant plasmid DNA solution, E. coli HB101 was transformedto obtain plasmid pBSMoSal shown in FIG. 30. Ten μg of the thus obtainedplasmid was reacted according to the recipe attached to AutoReadSequencing Kit (Pharmacia Biotech) and then electrophoresed with A.L.F.DNA Sequencer (Pharmacia Biotech) to thereby determine the basesequence. As a result, it was confirmed that the XhoI restriction sitelocated upstream of the promoter/enhancer of the terminal repeatedsequence of Moloney mouse leukemia virus had been eliminated.

Subsequently, 3 μg of the plasmid pBSMosal as obtained above were addedto 10 μl of a buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM magnesiumchloride and 1 mM DTT, to which 10 units of the restriction enzyme KpnI(Takara Shuzo) were added and reacted at 37° C. for 1 hour. The reactionmixture was ethanol-precipitated, and the precipitate was dissolved in10 μl of a buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM sodiumchloride, 10 mM magnesium chloride and 1 mM DTT, to which 10 units ofthe restriction enzyme HindIII (Takara Shuzo) were added and reacted at37° C. for 1 hour.

Then, the reaction mixture was subjected to agarose gel electrophoresisto thereby recover about 1 μg of an approx. 3.66 kb KpnI-HindIIIfragment. Subsequently, two synthetic DNAs having the base sequencesshown in SEQ ID NOS: 20 and 21, respectively, were synthesized using anautomatic DNA synthesizer (380A, Applied Biosystems). Then, 0.3 μg eachof the obtained synthetic DNAs were added to 15 μl of sterilized waterand heated at 65° C. for 5 minutes. The reaction mixture was left atroom temperature for 30 minutes. To this solution, 2 μl of a 10×buffer[500 mM Tris-HCl (pH 7.6), 100 mM magnesium chloride, 50 mM DTT] and 2μl of 10 mM ATP were added. Further, 10 units of T4 polynucleotidekinase (Takara Shuzo) were added and reacted at 37° C. for 30 minutes tophosphorylate the 5′ ends. Then, 0.1 μg of the KpnI-HindIII fragment(3.66 kb) from plasmid pBSMoSal as obtained above and 0.05 μg of thephosphorylated synthetic DNAs were added to sterilized water to give atotal volume of 20 μl and ligated using Ready-To-Go T4 DNA Ligase(Pharmacia Biotech). Using the thus obtained recombinant plasmid DNAsolution, E. coli HB101 was transformed to obtain plasmid pBSMoSalSshown in FIG. 31. Ten μg of the thus obtained plasmid were reactedaccording to the recipe attached to AutoRead Sequencing Kit (PharmaciaBiotech) and then electrophoresed with A.L.F. DNA Sequencer (PharmaciaBiotech) to thereby determine the base sequence. As a result, it wasconfirmed that the synthetic DNAs of interest had been transfected.

Subsequently, 10 μg of the plasmid pChiIgHB2 disclosed in Kokai No.304989/93 were dissolved in 10 μl of a buffer containing 50 mM Tris-HCl(pH 7.5), 100 mM sodium chloride, 10 mM magnesium chloride and 1 mM DTT,to which 10 units of the restriction enzyme Eco52I (Toyobo) were addedand reacted at 37° C. for 1 hour. The reaction mixture wasethanol-precipitated, and the precipitate was dissolved in 10 μl of abuffer containing 30 mM sodium acetate (pH 5.0), 100 mM sodium chloride,1 mM zinc acetate and 10% glycerol, to which 10 units of mung beannuclease (Takara Shuzo) were added and reacted at 37° C. for 10 minutes.The reaction mixture was subjected to phenol-chloroform extraction,followed by ethanol precipitation. Then, the sticky ends were bluntedusing DNA Blunting Kit (Takara Shuzo). After ethanol precipitation, theprecipitate was dissolved in 10 μl of a buffer containing 10 mM Tris-HCl(pH 7.5), 10 mM magnesium chloride and 1 mM DTT, to which 10 units ofthe restriction enzyme ApaI (Takara Shuzo) were added and reacted at 37°C. for 1 hour. The reaction mixture was subjected to agarose gelelectrophoresis to thereby recover about 0.7 μg of an approx. 0.99 kbApaI-blunt end fragment.

Subsequently, 3 μg of plasmid pBluescript SK(−) (Stratagene) were addedto 10 μl of a buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM magnesiumchloride and 1 mM DTT, to which 10 units of the restriction enzyme ApaI(Takara Shuzo) were added and reacted at 37° C. for 1 hour. The reactionmixture was ethanol-precipitated, and the precipitate was added to 10 μlof a buffer containing 33 mM Tris-HCl (pH 7.9), 10 mM magnesium acetate,66 mM potassium acetate, 0.5 mM DTT and 10 μg/ml BSA, to which 10 unitsof the restriction enzyme Smal (Takara Shuzo) were added and reacted at30° C. for 1 hour. The reaction mixture was subjected to agarose gelelectrophoresis to thereby recover about 1 μg of an approx. 3.0 kbApaI-Smal fragment.

Subsequently, 0.1 μg of the ApaI-blunt end fragment from plasmidpChilgHB2 as obtained above and 0.1 μg of the ApaI-Smal fragment frompBluescript SK(−) were added to sterilized water to give a total volumeof 20 μl and ligated using Ready-To-Go T4 DNA Ligase (PharmaciaBiotech). Using the thus obtained recombinant plasmid DNA solution, E.coli HB101 was transformed to obtain plasmid pBShCγ1 shown in FIG. 32.

Subsequently, 5 μg of the plasmid pBShCγ1 as obtained above were addedto 10 μl of a buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM magnesiumchloride and 1 mM DTT, to which 10 units of the restriction enzyme ApaI(Takara Shuzo) were added and reacted at 37° C. for 1 hour. The reactionmixture was ethanol-precipitated, and the precipitate was dissolved in10 μl of a buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM sodiumchloride, 10 mM magnesium chloride and 1 mM DTT, to which 10 units ofthe restriction enzyme SpeI (Takara Shuzo) were added and reacted at 37°C. for 1 hour. Then, the reaction mixture was subjected to agarose gelelectrophoresis to thereby recover about 1 μg of an approx. 1.0 kbApaI-SpeI fragment.

Subsequently, 3 μg of the plasmid pBSMoSalS as obtained above were addedto 10 μl of a buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM magnesiumchloride and 1 mM DTT, to which 10 units of the restriction enzyme ApaI(Takara Shuzo) were added and reacted at 37° C. for 1 hour. The reactionmixture was ethanol-precipitated, and the precipitate was dissolved in10 μl of a buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM sodiumchloride, 10 mM magnesium chloride and 1 mM DTT, to which 10 units ofthe restriction enzyme SpeI (Takara Shuzo) were added and reacted at 37°C. for 1 hour. Then, the reaction mixture was subjected to agarose gelelectrophoresis to thereby recover about 1 μg of an approx. 3.66 kbApaI-SpeI fragment.

Subsequently, 0.1 μg each of the ApaI-SpeI fragment from pBShCγ1 and theApaI-SpeI fragment from pBSMoSalS as obtained above were added tosterilized water to give a total volume of 20 μl and ligated usingReady-To-Go T4 DNA Ligase (Pharmacia Biotech). Using the thus obtainedrecombinant plasmid DNA solution, E. coli HB 101 was transformed toobtain plasmid pMohCγ1 shown in FIG. 33.

(6) Construction of Tandem Cassette-Type Humanized Antibody ExpressionVector pKANTEX93

A tandem cassette-type humanized antibody expression vector, pKANTEX93,was constructed as follows using the various plasmids obtained insubsections (1)–(5) of section 1 of Example 2.

Briefly, 3 μg of the plasmid pBSH-SAEE obtained in subsection (1) ofsection 1 of Example 2 were added to 10 μl of a buffer containing 10 mMTris-HCl (pH 7.5), 50 mM sodium chloride, 10 mM magnesium chloride and 1mM DTT, to which 10 units of the restriction enzyme HindIII (TakaraShuzo) were added and reacted at 37° C. for 1 hour. The reaction mixturewas ethanol-precipitated, and the precipitate was dissolved in 10 μl ofa buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 10mM magnesium chloride and 1 mM DTT, to which 10 units of the restrictionenzyme SalI (Takara Shuzo) were added and reacted at 37° C. for 1 hour.Then, the reaction mixture was subjected to agarose gel electrophoresisto thereby recover about 1 μg of an approx. 5.42 kb HindIII-SalIfragment.

Subsequently, 5 μg of the plasmid pBSK-HAEE obtained in subsection (1)of section 1 of Example 2 were added to 10 μl of a buffer containing 10mM Tris-HCl (pH 7.5), 50 mM sodium chloride, 10 mM magnesium chlorideand 1 mM DTT, to which 10 units of the restriction enzyme KpnI (TakaraShuzo) were added and reacted at 37° C. for 1 hour. The reaction mixturewas ethanol-precipitated, and the precipitate was dissolved in 10 μl ofa buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM sodium chloride, 10mM magnesium chloride and 1 mM DTT, to which 10 units of the restrictionenzyme HindIII (Takara Shuzo) were added and reacted at 37° C. for 1hour. Then, the reaction mixture was subjected to agarose gelelectrophoresis to thereby recover about 0.8 μg of an approx. 1.98 kbKpnI-HindIII fragment containing rabbit β-globin gene splicing poly (A)signal, SV40 early gene poly (A) signal and SV40 early gene promoter.

Subsequently, 5 μg of the plasmid pMohCγ1 obtained in subsection (5) ofsection 1 of Example 2 were added to 10 μl of a buffer containing 10 mMTris-HCl (pH 7.5), 10 mM magnesium chloride and 1 mM DTT, to which 10units of the restriction enzyme KpnI (Takara Shuzo) were added andreacted at 37° C. for 1 hour. The reaction mixture wasethanol-precipitated, and the precipitate was dissolved in 10 μl of abuffer containing 50 mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 10 mMmagnesium chloride and 1 mM DTT, to which 10 units of the restrictionenzyme SalI (Takara Shuzo) were added and reacted at 37° C. for 1 hour.Then, the reaction mixture was subjected to agarose gel electrophoresisto thereby recover about 0.8 μg of an approx. 1.66 kb KpnI-SalI fragmentcontaining the humanized antibody H chain expression unit.

Subsequently, 0.1 μg each of the HindIII-SalI fragment from pBSH-SAEE,the KpnI-HindIII fragment from pBSK-HAEE and the KpnI-SalI fragment frompMohCγ1 as obtained above were added to sterilized water to give a totalvolume of 20 μl and ligated using Ready-To-Go T4 DNA Ligase (PharmaciaBiotech). Using the thus obtained recombinant plasmid DNA solution, E.coli HB101 was transformed to obtain plasmid pMoγ1SP shown in FIG. 34.

Subsequently, 3 μg of the thus obtained plasmid pMoγ1SP were added to 10μl of a buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM sodiumchloride, 10 mM magnesium chloride and 1 mM DTT, to which 10 units eachof the restriction enzymes SalI (Takara Shuzo) and XhoI were added andreacted at 37° C. for 1 hour. The reaction mixture was subjected toagarose gel electrophoresis to thereby recover about 1 μg of an approx.9.06 kb SalI-XhoI fragment.

Subsequently, 5 μg of the plasmid pBSK-HAEESal obtained in subsection(2) of section 1 of Example 2 were added to 10 μl of a buffer containing10 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride and 1 mM DTT, to which10 units of the restriction enzyme KpnI (Takara Shuzo) were added andreacted at 37° C. for 1 hour. The reaction mixture wasethanol-precipitated, and the precipitate was dissolved in 10 μl of abuffer containing 50 mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 10 mMmagnesium chloride and 1 mM DTT, to which 10 units of the restrictionenzyme SalI (Takara Shuzo) were added and reacted at 37° C. for 1 hour.Then, the reaction mixture was subjected to agarose gel electrophoresisto thereby recover about 0.7 μg of an approx. 1.37 kb KpnI-SalI fragmentcontaining rabbit β-globin gene splicing signal, rabbit β-globin genesplicing signal poly (A) signal and SV40 early gene poly (A) signal.

Subsequently, 5 μg of the plasmid pMohCκ obtained in subsection (4) ofsection 1 of Example 2 were added to 10 μl of a buffer containing 10 mMTris-HCl (pH 7.5), 10 mM magnesium chloride and 1 mM DTT, to which 10units of the restriction enzyme KpnI (Takara Shuzo) were added andreacted at 37° C. for 1 hour. The reaction mixture wasethanol-precipitated, and the precipitate was dissolved in 10 μl of abuffer containing 50 mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 10 mMmagnesium chloride and 1 mM DTT, to which 10 units of the restrictionenzyme XhoI (Takara Shuzo) were added and reacted at 37° C. for 1 hour.Then, the reaction mixture was subjected to agarose gel electrophoresisto thereby recover about 0.7 μg of an approx. 1.06 kb KpnI-XhoI fragmentcontaining the humanized antibody L chain expression unit.

Subsequently, 0.1 μg each of the SalI-XhoI fragment from pMoγ1SP, theKpnI-SalI fragment from pBSK-HAEESal and the KpnI-XhoI fragment frompMohCκ as obtained above were added to sterilized water to give a totalvolume of 20 μl and ligated using Ready-To-Go T4 DNA Ligase (PharmaciaBiotech). Using the thus obtained recombinant plasmid DNA solution, E.coli HB101 was transformed to obtain plasmid pMoκγ1SP shown in FIG. 35.

Subsequently, 3 μg of the thus obtained plasmid pMoκγ1SP were dissolvedin 10 μl of a buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM sodiumchloride, 10 mM magnesium chloride and 1 mM DTT, to which 10 units ofthe restriction enzyme XhoI (Takara Shuzo) were added and reacted at 37°C. for 1 hour. The reaction mixture was ethanol-precipitated, and theprecipitate was added to 10 μl of a buffer containing 10 mM Tris-HCl (pH7.5), 10 mM magnesium chloride and 1 mM DTT, to which 10 units of therestriction enzyme SacII (Toyobo) were added and reacted at 37° C. for10 minutes so that the DNA fragments were partially digested. Then, thereaction mixture was subjected to agarose gel electrophoresis to therebyrecover about 0.2 μg of an approx. 8.49 kb SacII-XhoI fragment.

Subsequently, 3 μg of plasmid pBSX-SA obtained in subsection (3) ofsection 1 of Example 2 were added to 10 μl of a buffer containing 10 mMTris-HCl (pH 7.5), 10 mM magnesium chloride and 1 mM DTT, to which 10units of the restriction enzyme SacII (Toyobo) were added and reacted at37° C. for 1 hour. The reaction mixture was ethanol-precipitated, andthe precipitate was dissolved in 10 μl of a buffer containing 50 mMTris-HCl (pH 7.5), 100 mM sodium chloride, 10 mM magnesium chloride and1 mM DTT, to which 10 units of the restriction enzyme XhoI (TakaraShuzo) were added and reacted at 37° C. for 1 hour. Then, the reactionmixture was subjected to agarose gel electrophoresis to thereby recoverabout 1 μg of an approx. 4.25 kb SacII-XhoI fragment.

Subsequently, 0.1 μg each of the SacII-XhoI fragment from pMoκγ1SP andthe SacII-XhoI fragment from pBSX-SA as obtained above were added tosterilized water to give a total volume of 20 μl and ligated usingReady-To-Go T4 DNA Ligase (Pharmacia Biotech). Using the thus obtainedrecombinant plasmid DNA solution, E. coli HB101 was transformed toobtain plasmid pKANTEX93 shown in FIG. 36.

2. Isolation and Analysis of the cDNAs Coding for Anti-Human IL-5R αMonoclonal Antibodies

(1) Isolation of mRNA from Anti-Human IL-5R α MonoclonalAntibody-Producing Hybridomas

Using Fast Track, an mRNA extraction kit manufactured by Invitrogen,mRNA was isolated from 1×10⁸ cells each of mouse anti-human IL-5R αmonoclonal antibodies KM1257, KM1259 and KM1486 producing hybridoma celllines (corresponding to hybridomas FERM BP-5133, FERM BP-5134 and FERMBP-5651, respectively) in accordance with the instructions attached tothe kit.

(2) Preparation of H and L Chain cDNA Libraries from Mouse Anti-HumanIL-5R α Monoclonal Antibody-Producing Hybridomas

Using cDNA Synthesis Kit (Pharmacia Biotech) and according to theinstructions attached to the kit, a cDNA having an EcoRI adapter at bothends was synthesized separately from 5 μg each of the mRNAs obtainedfrom KM1257, KM1259 and KM1486 in subsection (1) of section 2 of Example2. About 6 μg of each cDNA were dissolved in 10 μl of sterilized waterand subjected to agarose gel electrophoresis, to thereby recover about0.1 μg each of an approx. 1.5 kb cDNA fragment corresponding to the cDNAencoding for the H chain of IgG type antibody and an approx. 1.0 kbfragment corresponding to the L chain of immunoglobulins. Then, 0.1 μgof the approx. 1.5 kb cDNA fragment or the approx. 1.0 kb cDNA fragmentand 1 μg of Lamda ZAPII vector (as treated with calf intestine alkalinephosphatase after cleavage with EcoRI; Stratagene) were dissolved in11.5 μl of T4 ligase buffer, to which 175 units of T4 DNA ligase wereadded and incubated at 12° C. for 24 hours, followed by incubation atroom temperature for another 2 hours. Using 4 μl of each reactionmixture, cDNAs were packed into a λ phage using Giga Pack Gold(Stratagene) by conventional methods (Molecular Cloning, 2.95, ColdSpring Harbor Laboratory, 1989). The resultant λ phages were infected toE. coli strain XL1-Blue [Biotechniques, 5, 376 (1987)] in Giga Pack Goldby conventional methods (Molecular Cloning, 2.95–107, Cold Spring HarborLaboratory, 1989) to obtain about 4000 phage clones for each of the Hchain cDNA library and the L chain cDNA library of KM1257, KM1259 andKM1486.

(3) Cloning of the cDNAs Coding for the H and L Chains of Anti-HumanIL-5R α Monoclonal Antibody-Producing Hybridomas

The recombinant phages prepared in subsection (2) of section 2 ofExample 2 was fixed on a nitrocellulose filter by conventional methods(Molecular Cloning, 2.12, Cold Spring Harbor Laboratory, 1989). The cDNAcoding for the C region of mouse Ig the H chain was a fragment frommouse Cγ1 cDNA [Cell, 18, 559 (1979)] and the L chain was a fragmentfrom mouse Cκ cDNA [Cell, 22, 197 (1980)] were labeled using ECL directnucleic acid labeling and detection systems (Amersham). Using thoselabeled cDNA as probes, recombinant phages were screened. Subsequently,according to the instructions attached to Lamda ZAPII vector(Stratagene), the phage clones were replaced with plasmidpBluescriptSK(−). Finally, the following plasmids were obtained:recombinant plasmid pKM1257H comprising a cDNA coding for the H chain ofKM1257 and recombinant plasmid pKM1257L comprising a cDNA coding for theL chain of KM1257; recombinant plasmid pKM1259H comprising a cDNA codingfor the H chain of KM1259 and recombinant plasmid pKM1259L comprising acDNA coding for the L chain of KM1259; and recombinant plasmid pKM1486Hcomprising a cDNA coding for the H chain of KM1486 and recombinantplasmid pKM1486L comprising a cDNA coding for the L chain of KM1486.

(4) Determination of the Base Sequences for the V Regions of the cDNAsCoding for the H and L Chains of Anti-Human IL-5R α MonoclonalAntibodies

The base sequence for the V region of each of the cDNAs coding for the Hand L chains of mouse anti-human IL-5R α monoclonal antibodies asobtained in subsection (3) of section 2 of Example 2 was analyzed byreacting 10 μg of the resultant plasmid according to the recipe attachedto AutoRead Sequencing Kit (Pharmacia Biotech) and then electrophoresedwith A.L.F. DNA Sequencer (Pharmacia Biotech). From the base sequencethus determined for each of the cDNAs, amino acid sequences for the Vregions of the L and H chains of KM1257, KM1259 and KM1486 weredetermined. SEQ ID NOS: 22 & 23 show the base sequence and amino acidsequence of the V region of the H chain of KM1257; SEQ ID NOS:24 & 25show those of the L chain of KM1257; SEQ ID NOS: 26 & 27 show those ofthe H chain of KM1259; SEQ ID NOS:28 & 29 show those of the L chain ofKM1259; SEQ ID NOS:30 & 31 show those of the H chain of KM1486; and SEQID NOS:32 & 33 show those of the L chain of KM1486.

(5) Identification of CDR sequences for the H and L Chains of Anti-HumanIL-5R α Monoclonal Antibodies

CDR sequence for each H chain and those for each L chain were identifiedfrom the amino acid sequences for the V regions of the H and L chains ofeach mouse anti-human IL-5R α as monoclonal antibody determined insubsection (4) of section 2 of Example 2 by comparing the above aminoacid sequences with the V region amino acid sequences for knownantibodies (Sequences of Proteins of Immunological Interest, US Dept.Health and Human Services, 1991). SEQ ID NOS: 34, 35 and 36 show theamino acid sequences for CDR1, CDR2 and CDR3, respectively, of the Hchain of KM1257. SEQ ID NOS: 37, 38 and 39 show the amino acid sequencesfor CDR1, CDR2 and CDR3, respectively, of the L chain of KM1257. SEQ IDNOS: 40, 41 and 42 show the amino acid sequences for CDR1, CDR2 andCDR3, respectively, of the H chain of KM1259. SEQ ID NOS: 43, 44 and 45show the amino acid sequences for CDR1, CDR2 and CDR3, respectively, ofthe L chain of KM1259. SEQ ID NOS: 46,47 and 48 show the amino acidsequences for CDR1, CDR2 and CDR3, respectively, of the H chain ofKM1486. SEQ ID NOS: 49, 50 and 51 show the amino acid sequences forCDR1, CDR2 and CDR3, respectively, of the L chain of KM1486.

3. Preparation of Anti-Human IL-5R α Human Chimeric Antibody

An anti-human IL-5R α human chimeric antibody derived from theanti-human IL-5R α monoclonal antibody KM1259 having an activity toinhibit the biological activity of human IL-5 was prepared as describedbelow.

(1) Construction of Expression Vector pKANTEX1259 for Anti-Human IL-5R αHuman Chimeric Antibody

An expression vector, pKANTEX1259, for an anti-human IL-5R α humanchimeric antibody was constructed as follows using the humanizedantibody expression vector pKANTEX93 constructed in section 1 of Example2 and the plasmids pKM1259H and pKM1259L obtained in section 2 ofExample 2.

Briefly, 3 μg of the humanized antibody expression vector pKANTEX93 wereadded to 10 μl of a buffer containing 10 mM Tris-HCl (pH 7.5), 10 mMmagnesium chloride and 1 mM DTT, to which 10 units of the restrictionenzyme ApaI (Takara Shuzo) were added and reacted at 37° C. for 1 hour.The reaction mixture was ethanol-precipitated, and the precipitate wasadded to 10 μl of a buffer containing 50 mM Tris-HCl (pH 7.5), 100 mMsodium chloride, 10 mM magnesium chloride, 1 mM DTT, 100 μg/ml BSA and0.01% Triton X-100, to which 10 units of the restriction enzyme NotI(Takara Shuzo) were added and reacted at 37° C. for 1 hour. The reactionmixture was subjected to agarose gel electrophoresis to thereby recoverabout 1 μg of an approx. 12.75 kb ApaI-NotI fragment. Subsequently, 5 μgof plasmid pKM1259H was added to 10 μl of a buffer containing 10 mMTris-HCl (pH 7.5), 10 mM magnesium chloride and 1 mM DTT, to which 10units of the restriction enzyme BanI (Toyobo) were added and reacted at37° C. for 1 hour. The reaction mixture was ethanol-precipitated, andthe precipitate was added to 10 μl of a buffer containing 50 mM Tris-HCl(pH 7.5), 100 mM sodium chloride, 10 mM magnesium chloride, 1 mM DTT,100 μg/ml BSA and 0.01% Triton X-100, to which 10 units of therestriction enzyme NotI (Takara Shuzo) were added and reacted at 37° C.for 1 hour. The reaction mixture was subjected to agarose gelelectrophoresis to thereby recover about 0.5 μg of an approx. 0.41 kbBanI-NotI fragment.

Subsequently, two synthetic DNAs having the base sequences shown in SEQID NOS: 52 and 53, respectively, were synthesized with an automatic DNAsynthesizer (380A, Applied Biosystems). Then, 0.3 μg each of theobtained synthetic DNAs were added to 15 μl of sterilized water andheated at 65° C. for 5 minutes. After the reaction mixture was left atroom temperature for 30 minutes, 21 μl of a 10×buffer [500 mM Tris-HCl(pH 7.6), 100 mM magnesium chloride, 50 mM DTT] and 2 μl of 10 mM ATPwere added. Further, 10 units of T4 polynucleotide kinase were added andreacted at 37° C. for 30 minutes to thereby phosphorylate the 5′ ends.

Then, 0.1 μg of the ApaI-NotI fragment from the humanized antibodyexpression vector pKANTEX93, 0.1 μg of the BanI-NotI fragment fromplasmid pKM1259H and 0.05 μg of the phosphorylated synthetic DNAs asobtained above were added to sterilized water to give a total volume of20 μl and ligated using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech).Using the thus obtained recombinant plasmid DNA solution, E. coli HB101was transformed to obtain plasmid pKANTEX1259H shown in FIG. 37.

Subsequently, 3 μg of the thus obtained plasmid pKANTEX1259H were addedto 10 μl of a buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM sodiumchloride, 10 mM magnesium chloride, 1 mM DTT and 100 μg/ml BSA, to which10 units each of the restriction enzymes EcoRI (Takara Shuzo) and SplI(Takara Shuzo) were added and reacted at 37° C. for 1 hour. The reactionmixture was subjected to agarose gel electrophoresis to thereby recoverabout 1 μg of an approx. 13.20 kb EcoRI-SplI fragment.

Subsequently, 5 μg of plasmid pKM1259L were added to 10 μl of a buffercontaining 10 mM Tris-HCl (pH 7.5), 50 mM sodium chloride, 10 mMmagnesium chloride and 1 mM DTT, to which 10 units of the restrictionenzyme AvaII (Takara Shuzo) were added and reacted at 37° C. for 1 hour.The reaction mixture was ethanol-precipitated, and the precipitate wasadded to 10 μl of a buffer containing 50 mM Tris-HCl (pH 7.5), 100 mMsodium chloride, 10 mM magnesium chloride and 1 mM DTT, to which 10units of the restriction enzyme EcoRI (Takara Shuzo) were added andreacted at 37° C. for 1 hour. The reaction mixture was subjected toagarose gel electrophoresis to thereby recover about 0.5 μg of anapprox. 0.38 kb AvaII-EcoRI fragment.

Subsequently, two synthetic DNAs having the base sequences shown in SEQID NOS: 54 and 55, respectively, were synthesized with an automatic DNAsynthesizer (380A, Applied Biosystems). Then, 0.3 μg each of theobtained synthetic DNAs were added to 15 μl of sterilized water andheated at 65° C. for 5 minutes. After the reaction mixture was left atroom temperature for 30 minutes, 2 μl of a 10×buffer [500 mM Tris-HCl(pH 7.6), 100 mM magnesium chloride, 50 mM DTT] and 2 μl of 10 mM ATPwere added. Further, 10 units of T4 polynucleotide kinase were added andreacted at 37° C. for 30 minutes to thereby phosphorylate the 5′ ends.

Then, 0.1 μg of the EcoRI-SplI fragment from plasmid KANTEX1259H, 0.1 μgof the AvaII-EcoRI fragment from plasmid pKM1259L and 0.05 μg of thephosphorylated synthetic DNAs as obtained above were added to sterilizedwater to give a total volume of 20 μl and ligated using Ready-To-Go T4DNA Ligase (Pharmacia Biotech). Using the thus obtained recombinantplasmid DNA solution, E. coli HB101 was transformed to obtain plasmidpKANTEX1259 shown in FIG. 38.

(2) Expression of Anti-Human IL-5 α Human Chimeric Antibody in RatMyeloma YB2/0 Cells (ATCC CRL1581) using pKANTEX1259

The transfection of the anti-human IL-5 α human chimeric antibodyexpression vector pKANTEX1259 into YB2/0 cells was performed accordingto the method of Miyaji et al. by electroporation [Cytotechnology,3,133, (1990)].

Briefly, 4 μg of the pKANTEX1259 obtained in subsection (1) of section 3of Example 2 were transfected into 4×10⁶ YB2/0 cells. Then,RPMI1640-FCS(10) was dispensed into a 96-well microtiter plate (200μl/well). Cells were cultured in a 5% CO₂ incubator at 37° C. for 24hours. Then, Geneticin (hereinafter referred to as “G418”; Gibco) wasadded to give a concentration of 0.5 mg/ml and cells were cultured foranother 1–2 weeks. The culture supernatants were recovered from thosewells which had become confluent with the appearance of transformantcolonies having G418 resistance. The activity of an anti-human IL-5R αhuman chimeric antibody in the supernatants was determined by ELISAmethod 1 or 2 as described below.

ELISA Method 1

The shIL-5R α-Fc obtained from the insect cell culture supernatant insubsection (10) of section 1 of Example 1 was diluted with PBS to aconcentration of 5 μg/ml or less. The diluent was dispensed into a96-well EIA plate (Greiner) (50 μl/well), which was left at 4° C.overnight to allow the protein to be adsorbed. After washing the plate,PBS containing 1% bovine serum albumin (BSA)(1% BSA-PBS) was added tothe plate in an amount of 100 μl/well and reacted at room temperaturefor 1 hour to thereby block the remaining active groups. Afterdiscarding 1% BSA-PBS, the culture supernatants from the transformant orvarious purified anti-human IL-5 α antibodies at a concentration of 40μg/ml were added to the plate in an amount of 25 μl/well. Further, thebiotin-labeled human IL-5 (0.4 μg/ml) prepared in section 3 of Example 1was added to the plate in an amount of 25 μl/well and reacted at roomtemperature for 4 hours. After washing with 0.05% Tween-PBS,peroxidase-labeled avidin D (Nippon Reizo) diluted 4000 folds with 1%BSA-PBS was added to the plate in an amount of 50 μl/well and reacted atroom temperature for 1 hour. After washing with 0.05% Tween-PBS, an ABTSsubstrate solution [as prepared by dissolving 550 mg of 2,2′azinobis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium in 1 L of 0.1M citrate buffer (pH 4.2) and adding 1 μl/ml of hydrogen peroxideimmediately before use] was added at 50 μl/well to allow colordevelopment. Then, the absorbance (OD) at 415 nm was measured. Theabsorbance value in the absence of an antibody was regarded as zeropercent inhibition, and the percent inhibitions of antibodies againstthe biotin-labeled IL-5 were calculated by the following formula toevaluate each sample.

${{Percent}\mspace{14mu}{binding}\mspace{14mu}{inhibition}} = {100 - {\frac{A - C}{B - C} \times 100}}$wherein

-   -   A: OD value in the presence of an antibody    -   B: OD value in the absence of an antibody    -   C: OD value in the absence of biotin-labeled human IL-5.        ELISA method 2

The shIL-5R α obtained from the insect cell culture supernatant insubsection (10) of section 1 of Example 1 was diluted with PBS to aconcentration of 2 μg/ml or less. The diluent was dispensed into a96-well EIA plate (Greiner) (50 μl/well), which was left at 4° C.overnight to allow the protein to be adsorbed. After washing the plate,PBS containing 1% bovine serum albumin (BSA)(1% BSA-PBS) was added tothe plate in an amount of 100 μl/well and reacted at room temperaturefor 1 hour to thereby block the remaining active groups. Afterdiscarding 1% BSA-PBS, the culture supernatants from the transformant orvarious purified anti-human IL-5 α antibodies at a concentration of 50μg/ml were added to the plate in an amount of 50 μl/well and reacted atroom temperature for 2 hours. After washing with 0.05% Tween-PBS,peroxidase-labeled anti-human IgG antibody (American QualexInternational, Inc.) diluted 500 folds with 1% BSA-PBS was added to theplate in an amount of 50 μl/well and reacted at room temperature for 1hour. After washing with 0.05% Tween-PBS, an ABTS substrate solution [asprepared by dissolving 550 mg of 2,2′azinobis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium in 1 L of 0.1M citrate buffer (pH 4.2) and adding 1 μl/ml of hydrogen peroxideimmediately before use] was added at 50 μl/well to allow colordevelopment. Then, the absorbance (OD) at 415 nm was measured.

Those transformants in which the activity of anti-human IL-5R α humanchimeric antibody was observed in their culture supernatants weresuspended in RPMI1640-FCS(10) medium containing 0.5 mg/ml G418 and 50 nMMTX (Sigma), and cultured in a 5% CO₂ incubator at 37° C. for 1–2 weeks,to thereby induce transformants having resistance to 50 nM MTX. Whentransformants became confluent in wells, the activity of anti-humanIL-5R α human chimeric antibody in the supernatant was measured byeither of the ELISA methods described above. Those transformants inwhich the activity was observed were further cultured in a mannersimilar to that described above, with the MTX concentration increased to100 nM and to 200 nM. Thus, transformants which could grow inRPMI1640-FCS(10) medium containing 0.5 mg/ml G418 and 200 nM MTX andwhich produced an anti-human IL-5R α human chimeric antibody wereobtained. The thus obtained transformants were subjected to cloning bythe applications of the limiting dilution method to thereby obtain finalanti-human IL-5R α human chimeric antibody-producing transformants. As aspecific example of the anti-human IL-5R α human chimericantibody-producing transformant, KM1399 (FERM BP-5650) may be given. Theanti-human IL-5R α human chimeric antibody produced by this strain wasdesignated as KM1399. The transformant KM1399 was deposited with theNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology on Sep. 3, 1996 under accession numberFERM BP-5650. The productivity of the anti-human IL-5R α human chimericantibody KM1399 in the transformant clone KM1399 was approximately 5μg/10⁶ cells/24 hr.

(3) Purification of the Anti-Human IL-5R α Human Chimeric AntibodyKM1399 from Culture Supernatant

The anti-human IL-5R α human chimeric antibody KM1399 obtained insubsection (2) of section 3 of Example 2 was suspended in GIT medium(Nippon Pharmaceuticals) containing 0.5 mg/ml G418 and 200 mM MTX togive a concentration of 1–2×10⁵ cells/ml, and dispensed in 200 mlportions into 175 cm² flasks (Greiner). The cells were cultured in a 5%CO₂ incubator at 37° C. for 5–7 days, and the culture supernatant wasrecovered when each flask became confluent. From about 1.0 liter of theculture supernatant, about 3 mg of purified anti-human IL-5R α humanchimeric antibody KM1399 were obtained using a Procep A (Bioprocessing)column. About 4 μg of the purified anti-human IL-5R α human chimericantibody KM1399 were electrophoresed according to known methods [Nature,227, 680 (1970)] to perform molecular weight analyses. The results areshown in FIG. 39. As seen from FIG. 39, the molecular weight of theantibody H chain was about 50 KDa and that of the antibody L chain about25 KDa under reducing conditions. Thus, the expression of the H and Lchains with correct molecular weights was confirmed. On the other hand,under non-reducing conditions, the molecular weight of the anti-humanIL-5R α human chimeric antibody KM1399 was about 140 KDa. Thus, theexpression of a human chimeric antibody of the correct molecule weightcomposed of two H chains and two L chains was confirmed. Further, the Nterminal amino acid sequences for the H and L chains of the purifiedanti-human IL-5R α human chimeric antibody KM1399 were analyzed with aprotein sequencer (470A, Applied Biosystems) by the automatic Edmanmethod. As a result, the expected correct amino acid sequences wereobtained.

(4) Reactivity of the Anti-Human IL-5R α Human Chimeric Antibody KM1399with Human IL-5R α (ELISA method 1)

The reactivities of the anti-human IL-5R α mouse antibody KM1259 and theanti-human IL-5R α human chimeric antibody KM1399 with human IL-5R αwere determined by the ELISA method 1 described in subsection (2) ofsection 3 of Example 2. The results are shown in FIG. 40. As seen fromFIG. 40, the anti-human IL-5R α human chimeric antibody KM1399 proved tohave a strong reactivity with human IL-5R α which was comparable to thereactivity of the anti-human IL-5R α mouse antibody KM1259.

4. Transient Expression of Anti-Human IL-5R α Human Chimeric Antibody inCOS-7 Cells (ATCC CRL1651)

In order to evaluate the activities of various versions of theanti-human IL-5R α human CDR-grafted antibody to be described later morequickly, the transient expression of an anti-human IL-5R α humanchimeric antibody in COS-7 cells was examined as follows usingpKANTEX1259 and a modified vector thereof by the lipofectamine method.

(1) Construction of a improved vector of pKANTEX1259

Since the efficiency of the transient expression of a gene in animalcells depends on the number of copies of the expression vectortransfected thereinto, it was assumed that a smaller expression vectorwould lead to a better expression efficiency. Therefore, a smalleranti-human IL-5R α human chimeric antibody expression vector, pT1259,was constructed as follows by deleting some regions of pKANTEX1259 whichwere believed not to influence the expression of an antibody.

Briefly, 3 μg of plasmid pKANTEX1259 were added to 10 μl of a buffercontaining 10 mM Tris-HCl (pH 7.5), 50 mM sodium chloride, 10 mMmagnesium chloride and 1 mM DTT, to which 10 units of the restrictionenzyme HindIII (Takara Shuzo) were added and reacted at 37° C. for 1hour. The reaction mixture was ethanol-precipitated and the precipitatewas added to 10 μl of a buffer containing 50 mM Tris-HCl (pH 7.5), 100mM sodium chloride, 10 mM magnesium chloride and 1 mM DTT, to which 10units of the restriction enzyme MluI (Takara Shuzo) were added andreacted at 37° C. for 1 hour. The reaction mixture wasethanol-precipitated and the 5′ sticky ends generated by the digestionwith the restriction enzyme were blunted using DNA Blunting Kit (TakaraShuzo). The reaction mixture was subjected to agarose gelelectrophoresis to thereby recover about 1 μg of an approx. 9.60 kb DNAfragment. Then, 0.1 μg of the recovered DNA fragment was added tosterilized water to give a total volume of 20 μl and ligated usingReady-To-Go T4 DNA Ligase (Pharmacia Biotech). Using the thus obtainedrecombinant plasmid DNA solution, E. coli HB101 was transformed tothereby recover plasmid pT1259 shown in FIG. 41.

(2) Transient Expression of Anti-Human IL-5R α Human Chimeric Antibodyusing pT1259

COS-7 cells at a concentration of 1×10⁵ cells/ml were dispersed into a6-well plate (2 ml/well) and cultured at 37° C. overnight. To 100 μl ofOPTI-MEM (Gibco), 2 μg of pT1259 were added, followed by addition of asolution obtained by adding 10 μl of lipofectamine reagent (Gibco) to100 μl of OPTI-MEM medium (Gibco). The resultant mixture was reacted atroom temperature for 40 minutes to thereby form a DNA-liposome complex.COS-7 cells described above were washed with 2 ml of OPTI-MEM medium(Gibco) twice, and the solution containing the DNA-liposome complex wasadded thereto. Then, the cells were cultured at 37° C. for 7 hours.After the removal of the cultured fluid, 2 ml of DMEM medium (Gibco)containing 10% FCS were added and the cells were cultured at 37° C. At72 hours from the start of the cultivation, the culture supernatant wasrecovered, and the activity of an anti-human IL-5R α human chimericantibody in the culture supernatant was evaluated by the ELISA method 1described in subsection (2) of section 3 of Example 2. As shown in FIG.42, concentration-dependent activity was observed in the culturesupernatant of COS-7 cells into which pT1259 had been transfected. Thus,the expression of an anti-human IL-5R α human chimeric antibody wasconfirmed. From these results, it has been shown to be possible toevaluate the activities of humanized antibodies derived from variousexpression vectors in a transient expression system by preparing aimproved small-size vector pKANTEX93, and by then transfecting thevector into COS-7 cells. Further, in order to compare correctly theactivities of the various anti-human IL-5R α human CDR-graftedantibodies to be described later, the concentration of antibody formedby the transient expression in culture supernatant was determined by theELISA method described in subsection (3) of section 4 below.

(3) Determination of the Humanized Antibody Concentration in theTransient Expression-Culture Supernatant by ELISA

To a 96-well microtiter plate, a solution obtained by diluting goatanti-human IgG(γ-chain) antibody (Institute of Medicine & Biology) to400 fold with PBS was dispensed (50 μl/well) and reacted at 4° C.overnight. After the removal of the antibody solution, 100 μl/well of 1%BSA-PBS were added and reacted at 37° C. for 1 hour to thereby block theremaining active groups. After discarding 1% BSA-PBS, 50 μl/well of thetransient expression-culture supernatant or the purified anti-humanIL-5R α human chimeric antibody KM1399 was added and reacted at roomtemperature for 1 hour. After the reaction, the mixture was removed andthe plate was washed with 0.05% Tween-PBS. Then, 50 μl/well of asolution obtained by diluting peroxidase-labeled mouse anti-humanκ Lchain antibody (Zymed) 500 folds with 1% BSA-PBS were added to the plateand reacted at room temperature for 1 hour. After washing with 0.05%Tween-PBS, 50 μl/well of ABTS substrate solution [as obtained bydissolving 550 mg of 2,2′azinobis(3-ethylbenzothiazoline-6-sulfonicacid)diammonium in 1 L of 0.1 M citrate buffer (pH 4.2) and adding 1μl/ml of hydrogen peroxide immediately before use] were added to allowcolor development. Then, the absorbance at OD of 415 nm was measured.

5. Preparation of an Anti-Human IL-5Rα Human CDR-Grafted Antibody

An anti-human IL-5R α human CDR-grafted antibody was prepared asdescribed below; the antibody had a comparable activity to the mouseanti-human IL-5R α monoclonal antibody KM1259 and the anti-human IL-5R αhuman chimeric antibody KM1399, both of which had an activity to inhibitthe biological activity of human IL-5.

(1) Construction of a cDNA coding for the VH of an Anti-Human IL-5R αHuman CDR-Grafted Antibody based on the Consensus Sequence for the VH ofKnown Human Antibodies

Kabat et al. (Sequences of Proteins of Immunological Interest, US Dept.Health and Human Services, 1991) classified various known human antibodyVH into subgroups 1-III (HSG I-III) based on the homology of FRsequence, and identified the consensus sequence for each subgroup. Thepresent inventors therefore decided to design an amino acid sequence foran anti-human IL-5R α human CDR-grafted antibody VH based on thoseconsensus sequences. First, in order to select a consensus sequence tobe used as the base, the homology between the FR sequence for the VH ofthe mouse anti-human IL-5R α monoclonal antibody KM1259 and the FRsequence of the consensus sequence of human antibody VH of each subgroupwas examined (Table 1).

TABLE 1 Homology (%) between the FR Sequence for Mouse KM1259VH and theFR Sequence of the Consensus Sequence of Human Antibody VH of EachSubgroup HSGI HSGII HSGIII 72.1 50.6 55.2

As a result, it was confirmed that mouse KM1259VH has the highesthomology to subgroup I in FR sequence. Thus, the amino acid sequence foran anti-human IL-5R α human CDR-grafted antibody VH was designed basedon the consensus sequence of subgroup I, and a cDNA coding for the aboveamino acid sequence was constructed as described below using PCR.

Briefly, 6 synthetic DNAs having the base sequences shown in SEQ ID NOS:56–61, respectively, were synthesized with an automatic DNA synthesizer(380A; Applied Biosystems). Each of the synthesized DNAs was added to 50μl of a buffer containing 10 mM Tris-HCl (pH 8.3), 50 mM potassiumchloride, 1.5 mM magnesium chloride, 0.001% gelatin, 200 μM dNTP, 0.5 μMM13primer RV (Takara Shuzo), 0.5 μM M13primer M4 (Takara Shuzo) and 2units of TaKaRa Taq DNA polymerase (Takara Shuzo) to give a finalconcentration of 0.1 μM. Then, the resultant mixture was covered with 50μl of mineral oil and set in a DNA thermal cycler (PJ480; Perkin Elmer).Then, PCR was performed through 30 cycles, each cycle consisting of 9°C. for 2 minutes, 55° C. for 2 minutes and 72° C. for 2 minutes. Thereaction mixture was ethanol-precipitated and the precipitate wasdissolved in 20 μl of TE buffer. Thereafter, the mixture was subjectedto agarose gel electrophoresis to thereby recover about 0.2 μg of anapprox. 0.48 kb amplified fragment.

Subsequently, 3 μg of plasmid pBluescriptSK(−) (Stratagene) were addedto 10 μl of a buffer containing 33 mM Tris-HCl (pH 7.9), 10 mM magnesiumacetate, 66 mM potassium acetate, 0.5 mM DTT and 100 μg/ml BSA, to which10 units of the restriction enzyme Smal (Takara Shuzo) were added andreacted at 30° C. for 1 hour. The reaction mixture wasethanol-precipitated, and the precipitate was added to 20 μl of a buffercontaining 50 mM Tris-HCl (pH 9.0) and 1 mM magnesium chloride, to which1 unit of alkaline phosphatase (E. coli C75, Takara Shuzo) was added andreacted at 37° C. for 1 hour to thereby dephosphorylate 5′ ends. Then,the reaction mixture was subjected to phenol-chloroform extraction,followed by ethanol precipitation. The precipitate was dissolved in 20μl of TE buffer.

Subsequently, 0.1 μg of the amplified fragment obtained by PCR and 0.1μg of the Smal fragment from pBluescriptSK(−) were added to sterilizedwater to give a total volume of 20 μl and ligated using Ready-To-Go T4DNA Ligase (Pharmacia Biotech). Using the thus obtained recombinantplasmid DNA solution, E. coli HB101 was transformed. From 10transformant clones, plasmid DNA was prepared individually and the basesequence thereof was determined. As a result, plasmid phKM1259HV0 shownin FIG. 43 comprising a cDNA coding for the amino acid sequence for ananti-human IL-5R α human CDR-grafted antibody VH of interest wasobtained. The base sequence and the amino acid sequence for theanti-human IL-5R α human CDR-grafted antibody VH contained inphKM1259HV0 (hereinafter referred to as “HV.0”) are shown in SEQ ID NOS:62 and 63.

(2) Construction of a cDNA coding for the VL of an Anti-Human IL-5RαHuman CDR-Grafted Antibody based on the Consensus Sequence for the VL ofKnown Human Antibodies

Kabat et al. classified various known human antibody VL into subgroups1-IV (HSG I-IV) based on the homology of FR sequence, and identified theconsensus sequence for each subgroup. The present inventors thereforedecided to design an amino acid sequence for an anti-human IL-5R α humanCDR-grafted antibody VL based on those consensus sequences. First, inorder to select a consensus sequence to be used as the base, thehomology between the FR sequence for the VH of the mouse anti-humanIL-5R α monoclonal antibody KM1259 and the FR sequence of the consensussequence of human antibody VL of each subgroup was examined (Table 2).

TABLE 2 Homology (%) between the FR Sequence for Mouse KM1259VL and theFR Sequence of the Consensus Sequence of Human Antibody VL of EachSubgroup HSGI HSGII HSGIII HSGIV 73.8 57.5 60.0 65.0

As a result, it was confirmed that mouse KM1259VL has the highesthomology to subgroup I in FR sequence. Thus, the amino acid sequence foran anti-human IL-5R α human CDR-grafted antibody VL was designed basedon the consensus sequence of subgroup I, and a cDNA coding for the aboveamino acid sequence was constructed as described below using PCR.

Briefly, 6 synthetic DNAs having the base sequences shown in SEQ ID NOS:64–69, respectively, were synthesized with an automatic DNA synthesizer(380A; Applied Biosystems). Each of the synthesized DNAs was added to 50μl of a buffer containing 10 mM Tris-HCl (pH 8.3), 50 mM potassiumchloride, 1.5 mM magnesium chloride, 0.001% gelatin, 200 μM dNTP, 0.5 μMM13primer RV (Takara Shuzo), 0.5 μM M13primer M4 (Takara Shuzo) and 2units of TaKaRa Taq DNA polymerase (Takara Shuzo) to give a finalconcentration of 0.1 μM. Then, the resultant mixture was covered with 50μl of mineral oil and set in a DNA thermal cycler (PJ480; Perkin Elmer).Then, PCR was performed through 30 cycles, each cycle consisting of 94°C. for 2 minutes, 55° C. for 2 minutes and 72° C. for 2 minutes. Thereaction mixture was ethanol-precipitated and the precipitate wasdissolved in 20 μl of TE buffer. Thereafter, the solution was subjectedto agarose gel electrophoresis to thereby recover about 0.2 μg of anapprox. 0.43 kb amplified fragment.

Subsequently, 0.1 μg of the amplified fragment obtained above by PCR and0.1 μg of the Smal fragment from pBluescriptSK(−) obtained in subsection(1) of section 5 of Example 2 were added to sterilized water to give atotal volume of 20 μl and ligated using Ready-To-Go T4 DNA Ligase(Pharmacia Biotech). Using the thus obtained recombinant plasmid DNAsolution, E. coli HB101 was transformed. From 10 transformant clones,plasmid DNA was prepared individually and the base sequence thereof wasdetermined. As a result, plasmid phKM1259LV0 shown in FIG. 44 comprisinga cDNA coding for the amino acid sequence for the anti-human IL-5R αhuman CDR-grafted antibody VL of interest was obtained. The basesequence and the amino acid sequence for the anti-human IL-5R α humanCDR-grafted antibody VL contained in phKM1259LV0 (hereinafter referredto as “LV.0”) are shown in SEQ ID NOS: 70 & 71.

(3) Construction of Expression Vector for Anti-Human IL-5R α HumanCDR-Grafted Antibody pKANTEX1259HV0LV0, Based on the Consensus Sequenceof V Regions of Known Human Antibodies

An anti-human IL-5R α human CDR-grafted antibody expression vector,pKANTEX1259HV0LV0, was constructed as described below using thehumanized antibody expression vector pKANTEX93 obtained in section 1 ofExample 2, the plasmid phKM1259HV0 obtained in subsection (1) of section5 of Example 2 and the plasmid phKM1259LV0 obtained in subsection (2) ofsection 5 of Example 2.

Briefly, 5 μg of plasmid pKMh1259HV0 were added to 10 μl of a buffercontaining 10 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride and 1 mMDTT, to which 10 units of the restriction enzyme ApaI (Takara Shuzo)were added and reacted at 37° C. for 1 hour. The reaction mixture wasethanol-precipitated and the precipitate was added to 10 μl of a buffercontaining 50 mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 10 mMmagnesium chloride, 1 mM DTT, 100 μg/ml BSA and 0.01% Triton X-100, towhich 10 units of the restriction enzyme NotI (Takara Shuzo) were addedand reacted at 37° C. for 1 hour. The reaction mixture was subjected toagarose gel electrophoresis to thereby recover about 0.5 μg of anapprox. 0.44 kb ApaI-NotI fragment.

Subsequently, 0.1 μg of the ApaI-NotI fragment from the humanizedantibody expression vector pKANTEX93 obtained in subsection (1) ofsection 3 of Example 2 and 0.1 μg of the ApaI-NotI fragment from plasmidphKM1259HV0 obtained above were added to sterilized water to give atotal volume of 20 μl and ligated using Ready-To-Go T4 DNA Ligase(Pharmacia Biotech). Using the thus obtained recombinant plasmid DNAsolution, E. coli HB101 was transformed to thereby obtain plasmidpKANTEX1259HV0 shown in FIG. 45.

Subsequently, 3 μg of the thus obtained plasmid pKANTEX1259HV0 wereadded to 10 μl of a buffer containing 50 mM Tris-HCl (pH 7.5), 100 mMsodium chloride, 10 mM magnesium chloride, 1 mM DTT and 100 μg/ml BSA,to which 10 units each of the restriction enzyme EcoRI (Takara Shuzo)and the restriction enzyme SplI (Takara Shuzo) were added and reacted at37° C. for 1 hour. The reaction mixture was subjected to agarose gelelectrophoresis to thereby recover about 1 μg of an approx. 13.20 kbEcoRI-SplI fragment.

Subsequently, 5 μg of plasmid phKM1259LV0 were added to 10 μl of abuffer containing 50 mM Tris-HCl (pH 7.5), 100 mM sodium chloride, 10 mMmagnesium chloride, 1 mM DTT and 100 μg/ml BSA, to which 10 units eachof the restriction enzyme EcoRI (Takara Shuzo) and the restrictionenzyme SplI (Takara Shuzo) were added and reacted at 37° C. for 1 hour.The reaction mixture was subjected to agarose gel electrophoresis tothereby recover about 0.5 μg of an approx. 0.39 kb EcoRI-SplI fragment.

Then, 0.1 μg of the EcoRI-SplI fragment from plasmid pKANTEX1259HV0obtained above and 0.1 μg of the EcoRI-SplI fragment from plasmidphKM1259LV0 obtained above were added to sterilized water to give atotal volume of 20 μl and ligated using Ready-To-Go T4 DNA Ligase(Pharmacia Biotech). Using the thus obtained recombinant plasmid DNAsolution, E. coli HB101 was transformed to thereby obtain plasmidpKANTEX1259HV0LV0 shown in FIG. 46.

(4) Expression of an Anti-Human IL-5R α Human CDR-Grafted Antibody Basedon the Consensus Sequence of Known Human Antibody V Regions in RatMyeloma YB2/0 Cells (ATCC CRL1581) using pKANTEX1259HV0LV0

The expression of an anti-human IL-5R α human CDR-grafted antibody basedon the consensus sequence of known human antibody V regions in ratmyeloma YB2/0 cells (ATCC CRL1581) was performed using pKANTEX1259HV0LV0according to the method described in subsection (2) of section 3 ofExample 2.

As a result, KM8397 was obtained as a transformant producing ananti-human IL-5R α human CDR-grafted antibody based on the consensussequence of known human antibody V regions. The anti-human IL-5R α humanCDR-grafted antibody produced by the strain was designated as KM8397.The productivity of the anti-human IL-5R α human CDR-grafted antibodyKM8397 in the transformant KM8397 was about 4 μg/10⁶ cells/24 hr.

(5) Purification of the Anti-Human IL-5R α Human CDR-Grafted AntibodyKM8397 from Culture Supernatant

The anti-human IL-5R α human CDR-grafted antibody-producing clone KM8397obtained in subsection (4) of section 5 of Example 2 was culturedaccording to the method described in subsection (3) of section 3 ofExample 2 and purified to thereby obtain about 2 mg of KM8397. About 4μg of the purified anti-human IL-5R α human CDR-grafted antibody KM8397was electrophoresed according to the method described in subsection (3)of section 3 of Example 2 in order to examine its molecular weight. Theresults are shown in FIG. 47. As shown in FIG. 47, the molecular weightof the antibody H chain is about 50 KDa and that of the antibody L chainabout 25 KDa under reducing conditions. Thus, the expression of the Hand L chains with the correct molecular weights was confirmed. On theother hand, under non-reducing conditions, the molecular weight of theanti-human IL-5R α human CDR-grafted antibody KM8397 is about 140 KDa.Thus, the expression of a human CDR-grafted antibody of the correct sizecomposed of two H chains and two L chains was confirmed. Further, the Nterminal amino acid sequences for the H and L chains of the purifiedanti-human IL-5R α human CDR-grafted antibody KM8397 were analyzed witha protein sequencer (470A, Applied Biosystems) by the automatic Edmanmethod. As a result, the correct amino acid sequences as expected wereobtained.

(6) Reactivity of the Anti-Human IL-5R α Human CDR-Grafted AntibodyKM8397 with Human IL-5R α (ELISA method 2)

The reactivities of the anti-human IL-5R α human chimeric antibodyKM1399 and the anti-human IL-5R α human CDR-grafted antibody KM8397 withhuman IL-5R α were determined by the ELISA method 2 described insubsection (2) of section 3 of Example 2. The results are shown in FIG.48. As shown in FIG. 48, the reactivity of the anti-human IL-5R α humanCDR-grafted antibody KM8397 with human IL-5R α was shown to be about onehalf the reactivity of the anti-human IL-5R α human chimeric antibodyKM1399.

6. Increase in Activity by Modification of the Amino Acid Sequence forthe V Region of the Anti-Human IL-5R α Human CDR-Grafted Antibody KM8397

The reactivity of the anti-human IL-5R α human CDR-grafted antibodyKM8397 with human IL-5R α decreased to about one half the reactivity ofthe anti-human IL-5R α human chimeric antibody KM1399.

Therefore, the activity of KM8397 was increased by modifying the aminoacid sequence for the V region thereof by the methods described below.

(1) Modification of the Amino Acid Sequence for VH of the Anti-HumanIL-5R α Human CDR-Grafted Antibody KM8397

By mutating the amino acids of VH of the anti-human IL-5R α humanCDR-grafted antibody KM8397 shown in SEQ ID NOS: 62 & 63, variousmodified versions of VH of the anti-human IL-5R α human CDR-graftedantibody were prepared. The amino acids to be mutated were selected atrandom with reference to a computerized three-dimensional structuralmodel for the V region of the anti-human IL-5R α mouse antibody KM1259.As the method for transfecting a mutation, a plasmid comprising a cDNAcoding for a modified version of VH of interest of the anti-human IL-5Rα human CDR-grafted antibody was obtained by performing the proceduresdescribed in subsection (1) of section 5 of Example 2 using primers formutation.

Actually, a plasmid, phKM1259HV1, comprising a cDNA coding for themodified version 1 of VH (hereinafter referred to as “HV.1”) of theanti-human IL-5R α human CDR-grafted antibody shown in SEQ ID NOS: 73 &74 was obtained by performing the procedures described in subsection (1)of section 5 of Example 2 using the sequence shown in SEQ ID NO: 72 as aprimer for mutation and using synthetic DNAs having base sequences ofSEQ ID NOS: 56,57,58,59,72 and 61, respectively. In the amino acidsequence of HV.1, tyrosine in position 95 and alanine in position 97located in the FR of SEQ ID NOS:62 and 63 have been replaced withleucine and glycine, respectively, which are the amino acids found inthe V region of the mouse antibody KM1259H chain and this is in order toretain the reactivity with human IL-5R α recognized in the mouseantibody and the human chimeric antibody.

Further, a plasmid, phKM1259HV2, comprising a cDNA coding for themodified version 2 of VH (hereinafter referred to as “HV.2”) of theanti-human IL-5R α human CDR-grafted antibody shown in SEQ ID NOS:77 &78 was obtained by performing the procedures described in subsection (1)of section 5 of Example 2 using the sequences shown in SEQ ID NOS: 72,75 and 76 as primers for mutation and using synthetic DNAs having basesequences of SEQ ID NOS: 56, 57, 75, 76, 72 and 61, respectively. In theamino acid sequence of HV.2, glutamic acid in position 46, threonine inposition 74, tyrosine in position 95 and alanine in position 97 locatedin the FR of SEQ ID NO: 63 have been replaced with alanine, arginine,leucine and glycine, respectively, which are the amino acids found inthe V region of the mouse antibody KM1259H chain and this is in order toretain the reactivity with human IL-5R α recognized in the mouseantibody and the human chimeric antibody.

Further, a plasmid, phKM1259HV3, comprising a cDNA coding for themodified version 3 of VH (hereinafter referred to as “HV.3”) of theanti-human IL-5R α human CDR-grafted antibody shown in SEQ ID NOS: 82 &83 was obtained by performing the procedures described in subsection (1)of section 5 of Example 2 using the sequences shown in SEQ ID NOS: 79,80 and 81 as primers for mutation and using synthetic DNAs having basesequences of SEQ ID NOS: 56, 57, 79, 80, 81 and 61, respectively. In theamino acid sequence of HV.3, alanine in position 40, glutamic acid inposition 46, arginine in position 67, alanine in position 72, threoninein position 74, alanine in position 79, tyrosine in position 95 andalanine in position 97 located in the FR of SEQ ID NO: 63 have beenreplaced with arginine, alanine, lysine, serine, arginine, valine,leucine and glycine, respectively, which are the amino acids found inthe V region of the mouse antibody KM1259H chain and this is in order toretain the reactivity with human IL-5R a recognized in the mouseantibody and the human chimeric antibody.

As version advances from HV.0 to HV.4 one by one, the number of themonoclonal antibody-derived amino acids involved in the modificationincreases with increasing version number from HV.0 to HV.3.

(2) Modification of the Amino Acid Sequence for VL of the Anti-HumanIL-5R α Human CDR-Grafted Antibody KM8397

By mutating the amino acids of VL of the anti-human IL-5R α humanCDR-grafted antibody KM8397 shown in SEQ ID NO: 71, various modifiedversions of VL of the anti-human IL-5R α human CDR-grafted antibody wereprepared. The amino acids to be mutated were selected at random withreference to a computerized 3D structural model for the V region of theanti-human IL-5R α antibody KM1259. As the method for transfecting amutation, a plasmid comprising a cDNA coding for a modified version ofVL of interest of the anti-human IL-5R α human CDR-grafted antibody wasobtained by performing the procedures described in subsection (1) ofsection 5 of Example 2 using primers for mutation.

Actually, a plasmid, phKM1259LV1, comprising a cDNA coding for themodified version 1 of VL (hereinafter referred to as “LV.1”) of theanti-human IL-5R α human CDR-grafted antibody shown in SEQ ID NOS: 87 &88 was obtained by performing the procedures described in subsection (1)of section 5 of Example 2 using the sequences shown in SEQ ID NO: 84, 85and 86 as primers for mutation and using synthetic DNAs having basesequences of SEQ ID NOS: 64, 65, 84, 85, 68 and 86, respectively. In theamino acid sequence of LV.1, glutamine in position 37, lysine inposition 45 and phenylalanine in position 98 located in the FR of SEQ IDNO: 71 have been replaced with arginine, glutamic acid and valine,respectively, which are the amino acids found in the V region of themonoclonal antibody KM1259 L chain and this is in order to retain thereactivity with human IL-5R α recognized in the monoclonal antibody andthe human chimeric antibody.

Further, a plasmid, phKM1259LV2, comprising a cDNA coding for themodified version 2 of VL (hereinafter referred to as “LV.2”) of theanti-human IL-5R α human CDR-grafted antibody shown in SEQ ID NOS: 91 &92 was obtained by performing the procedures described in subsection (1)of section 5 of Example 2 using the sequences shown in SEQ ID NOS: 85,86, 89 and 90 as primers for mutation and using synthetic DNAs havingbase sequences of SEQ ID NOS: 64, 65, 89, 85, 90 and 86, respectively.In the amino acid sequence for LV.2, threonine in position 22, glutaminein position 37, lysine in position 45, serine in position 77 andphenylalanine in position 98 located in the FR of SEQ ID NO: 71 havebeen replaced with glycine, arginine, glutamic acid, aspartic acid andvaline, respectively, which are the amino acids found in the V region ofthe monoclonal antibody KM1259 L chain and this is in order to retainthe reactivity with human IL-5R α recognized in the monoclonal antibodyand the human chimeric antibody.

Further, a plasmid, phKM1259LV3, comprising a cDNA coding for themodified version 3 of VL (hereinafter referred to as “LV.3”) of theanti-human IL-5R α human CDR-grafted antibody shown in SEQ ID NOS: 97 &98 was obtained by performing the procedures described in subsection (1)of section 5 of Example 2 using the sequences shown in SEQ ID NOS: 85,93, 94, 95 and 96 as primers for mutation and using synthetic DNAshaving base sequences of SEQ ID NOS: 64, 93, 94, 85, 95 and 96,respectively. In the amino acid sequence of LV.3, serine in position 7,proline in position 8, threonine in position 22, glutamine in position37, glutamine in position 38, lysine in position 45, serine in position77, tyrosine in position 87 and phenylalanine in position 98 located inthe FR of SEQ ID NO: 71 have been replaced with alanine, threonine,glycine, arginine, lysine, glutamic acid, aspartic acid, phenylalanineand valine, respectively, which are the amino acids found in the Vregion of the monoclonal antibody KM1259 L chain and this is in order toretain the reactivity with human IL-5R α recognized in the monoclonalantibody and the human chimeric antibody.

Further, a plasmid, phKM1259LV4, comprising a cDNA coding for themodified version 4 of VL (hereinafter referred to as “LV.4”) of theanti-human IL-5R α human CDR-grafted antibody shown in SEQ ID NOS: 102and 103 was obtained by performing the procedures described insubsection (1) of section 5 of Example 2 using the sequences shown inSEQ ID NOS: 93, 96, 99, 100 and 101 as primers for mutation and usingsynthetic DNAs having base sequences of SEQ ID NOS: 64, 93, 99, 100, 101and 96, respectively. In the amino acid sequence of LV.4, serine inposition 7, proline in position 8, threonine in position 22, glutaminein position 37, glutamine in position 38, proline in position 44, lysinein position 45, phenylalanine in position 71, serine in position 77,tyrosine in position 87 and phenylalanine in position 98 located in theFR of SEQ ID NO: 71 have been replaced with alanine, threonine, glycine,arginine, lysine, valine, glutamic acid, tyrosine, aspartic acid,phenylalanine and valine, respectively, which are the amino acids foundin the V region of the monoclonal antibody KM1259 L chain and this is inorder to retain the reactivity with human IL-5R α recognized in themonoclonal antibody and the human chimeric antibody.

As a result, as version advances from LV.0 to HV.4 one by one, thenumber of the monoclonal antibody-derived amino acids involved in themodification increases with increasing version number from LV.0 to LV.4.

(3) Preparation of Anti-Human IL-5R α Human CDR-Grafted AntibodiesHaving Various Modified Versions of V Region

Using the humanized antibody expression vector pKANTEX93 constructed insection 1 of Example 2 and the various plasmids comprising cDNAs codingfor various modified versions of the V region of the anti-human IL-5R αhuman CDR-grafted antibody obtained in subsections (1) and (2) ofsection 5 of Example 2, vectors for the expression of anti-human IL-5R αhuman CDR-grafted antibodies having various modified versions of the Vregion were constructed by the method described in subsection (3) ofsection 5 of Example 2. Table 3 shows combinations of various modifiedversions of the V region used in the expression vectors constructed andthe designation of these expression vectors.

Among these expression vectors, a total of 13 vectors pKANTEX1259HV0LV0,pKANTEX1259HV1LV0, pKANTEX1259HV2LV0, pKANTEX1259HV0LV1,pKANTEX1259HV1LV1, pKANTEX1259HV2LV1, pKANTEX1259HV0LV2,pKANTEX1259HV1LV2, pKANTEX1259HV2LV2, pKANTEX1259HV0LV3,pKANTEX1259HV2LV3, pKANTEX1259HV2LV3, and pKANTEX1259HV3LV3 weremodified into transient expression vectors by the method described insubsection (1) of section 4 of Example 2. Using these transientexpression vectors and in accordance with the method described insubsection (2) of section 4 of Example 2, the transient expression ofanti-human IL-5R α human CDR-grafted antibodies having various modifiedversions of the V region was performed. As a control, the transientexpression of the anti-human IL-5R α human chimeric antibody KM1399 wasperformed simultaneously. The binding activity for human IL-5R α of anantibody in the culture supernatant was determined by the ELISA method 1described in subsection (2) of section 3 of Example 2, and the antibodyconcentration in the culture supernatant was determined by the ELISAmethod described in subsection (3) of section 4 of Example 2. Using twoELISA methods, the activities of anti-human IL-5R α human CDR-graftedantibodies having various modified versions of the V region are shown inFIG. 49 as relative values in which the activity of the human chimericantibody KM1399 is taken as 100. In FIG. 49, various modified versionsof anti-human IL-5R α human CDR-grafted antibodies are represented by acombination of VH and VL. From FIG. 49, a tendency is recognized with VHsuch that the activity increases as modification proceeds from HV.0 toHV.3. With respect to VL, a tendency is recognized such that thereactivity is high in LV.0 and LV.3 but low in LV.1 and LV.2. Then, amore accurate activity evaluation of anti-human IL-5R α humanCDR-grafted antibodies comprising combinations of LV.0 and variousmodified VH; LV.3 and HV.0; LV.3 and HV.3; and LV.4 which is a furthermodified version of LV.3, and various modified VH was performed usingpurified antibodies as follows.

Briefly, using the 10 expression vectors for anti-human IL-5R α humanCDR-grafted antibodies described above, i.e., pKANTEX1259HV0LV0,pKANTEX1259HV1LV0, pKANTEX1259HV2LV0, pKANTEX1259HV3LV0,pKANTEX1259HV0LV3, pKANTEX1259HV3LV3, pKANTEX1259HV0LV4,pKANTEX1259HV1LV4, pKANTEX1259HV2LV4, and pKANTEX1259HV3LV4 andaccording to the method described in subsection (2) of section 3 ofExample 2, antibodies of interest were expressed in YB2/0 cells tothereby obtain transformant producing various anti-human IL-5R α humanCDR-grafted antibodies at a productivity level of 2–4 μg/10⁶ cells/24hr. The transformants producing various anti-human IL-5R α humanCDR-grafted antibodies were cultured and purified by the methodsdescribed in subsection (3) of section 3 of Example 2 to thereby obtain1–2 mg each of various anti-human IL-5R α human CDR-grafted antibodies.About 4 μg each of the various purified anti-human IL-5R α humanCDR-grafted antibodies were electrophoresed by the method described insubsection (3) of section 3 of Example 2 to measure their molecularweights. Under reducing conditions, the molecular weight of the antibodyH chain is about 50 kDa and that of the antibody L chain about 25 kDa ineach of the anti-human IL-5R α human CDR-grafted antibodies. Thus, theexpression of H and L chains with the correct molecular weights wasconfirmed. Under non-reducing conditions, the molecular weight of theantibody is about 140 kDa in each of the anti-human IL-5R α humanCDR-grafted antibodies. Thus, the expression of human CDR-graftedantibodies each composed of two H chains and two L chains of the correctsize was confirmed. Further, the N terminal amino acid sequences for theH and L chains of the various purified anti-human IL-5R α humanCDR-grafted antibodies were analyzed with a protein sequencer (470A,Applied Biosystems) by the automatic Edman method. As a result, thecorrect amino acid sequences as expected were obtained in each of thoseantibodies.

The reactivity with human IL-5R α in the various purified anti-humanIL-5R α human CDR-grafted antibodies obtained above was determined bythe ELISA method 2 described in subsection (2) of section 3 of Example 2and the results are shown in FIG. 50. In FIG. 50, various modifiedversions of anti-human IL-5R α human CDR-grafted antibodies arerepresented by a combination of VH and VL. As shown in FIG. 50, of the10 purified anti-human IL-5R α human CDR-grafted antibodies, HV.3LV.0and HV.3LV.4 proved to have a reactivity with human IL-5R α as strong asthe reactivity of the anti-human IL-5R α human chimeric antibody KM1399.

When the amino acid sequences for the anti-human IL-5R α humanCDR-grafted antibodies HV.3LV.0 and HV.3LV.4 exhibiting a reactivitywith human IL-5R α as strong as the reactivity of the anti-human IL-5R αhuman chimeric antibody KM1399 are compared, both have the same aminoacid sequence which is shown as HV.3 for VH but they have differentamino acid sequences for VL, i.e., shown as LV.0 and LV.4. While LV.0 isa sequence obtained by simply grafting the CDR to the FR of a humanantibody, LV.4 is a sequence obtained by converting 11 amino acidresidues within the FR of a human antibody to those amino acid residuesfound in the monoclonal antibody in order to increase activity. However,from the results shown in FIG. 50, the modification of amino acidresidues makes little contributions to the increase of activityactually. Based on these facts, HV.3LV.0 which has a reactivity withhuman IL-5Rα as strong as the reactivity of the anti-human IL-5R α humanchimeric antibody KM1399 and which is expected to be less antigenicagainst humans since the replacement of amino acids derived from themonoclonal antibody is less, has been selected as an anti-human IL-5R αhuman CDR-grafted antibody. HV.3LV.0 was designated as KM8399, and thetransformant KM8399 producing the anti-human IL-5Rα human CDR-graftedantibody KM8399 was deposited with the National Institute of Bioscienceand Human-Technology, Agency of Industrial Science and Technology onSep. 3, 1996 under accession number FERM BP-5648.

In the preparation of the anti-human IL-5R α human CDR-grafted antibodyKM8399, the following matters have been taken into consideration. Asseen in the preparation of other human CDR-grafted antibodies, theactivity in the anti-human IL-5R α human CDR-grafted antibody KM8397,which was obtained by simply grafting only the CDR of the anti-humanIL-5R α monoclonal antibody KM1259 into the FR of a human antibody,decreased to about ½ of the activity of the monoclonal antibody KM1259.Hence, several amino acids within the FR of the V regions of H and Lchains were modified into the amino acids found in the monoclonalantibody KM1259, and examined for an increase in activity. With respectto VH, the activity increased as the modification proceeded. On theother hand, with respect to VL, the modification of a small number ofamino acids resulted in a decrease in activity; although the activitycan be increased by increasing the number of amino acids modified, theactivity only rose to the level of unmodified VL. Although the cause ofthis fact cannot be completely clarified without more detailed analysis(e.g., X-ray crystal analysis), the interaction between the VH and VL ofan antibody is probably involved and the results of such interactionwould vary depending on the antibody used. Because of such problems, noefficient method has yet been established for preparing a humanCDR-grafted antibody of which is applicable to any antibody and trialsand errors as made in the present Example are required. With such trialsand errors being accumulated, a more efficient method for preparinghuman CDR-grafted antibodies could be established. The present Exampleshows the first case of successful preparation of an anti-human IL-5R αhuman CDR-grafted antibody and thus provides suggestions for efficientpreparation of human CDR-grafted antibodies.

7. Preparation of Anti-Human IL-5R α Humanized Antibodies of HumanAntibody IgG4 Subclass

(1) Isolation and Analysis of a cDNA coding for the C Region (Cγ4) ofHuman Antibody IgG4 Subclass

1.1×10⁷ B cells were separated from 200 ml of peripheral blood from ahealthy volunteer using anti-CD19 antibody coated Dynabeads (DYNABEADSM-450 Pan-B(CD19); Nippon Dyner) and DETACHaBEAD (Nippon Dyner) inaccordance with the attached instructions. Then, mRNA was obtained fromthe separated cells using Quick Prep mRNA Purification Kit (PharmaciaBiotech) in accordance with the attached instructions. From all of themRNA obtained, cDNA was synthesized using Time Saver cDNA Synthesis Kit(Pharmacia Biotech) in accordance with the attached instructions. ThenPCR was performed as described in subsection (1) of section 5 of Example2 using all of the cDNA obtained above and using, as primers, syntheticDNAs shown in SEQ ID NOS: 104 and 105 which are homologous to the 5′ and3′ sides of a cDNA coding for human antibody Cγ4 [Nucleic Acid Research,14, 1789 (1986)]. The 5′ side and 3′ side primers used in the PCR hadbeen designed to have recognition sequences for the restriction enzymesApaI and BamHI at their 5′ terminals so that the cDNA to be obtainedcould be easily inserted into a humanized antibody expression vector.The reaction mixture after the PCR was purified with QIAquick PCRPurification Kit (Qiagen) and then added to 30 μl of a buffer containing10 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride and 1 mM DTT. To theresultant mixture, 10 units of the restriction enzyme ApaI (TakaraShuzo) were added and reacted at 37° C. for 1 hour. The reaction mixturewas ethanol-precipitated and the precipitate was added to 10 μl of abuffer containing 20 mM Tris-HCl (pH 8.5), 100 mM potassium chloride, 10mM magnesium chloride and 1 mM DTT, to which 10 units of the restrictionenzyme BamHI (Takara Shuzo) were added and reacted at 30° C. for 1 hour.The reaction mixture was subjected to agarose gel electrophoresis tothereby recover about 0.5 μg of an approx. 1.0 kb ApaI-BamHI fragment.

Subsequently, 3 μg of plasmid pBluescriptSK(−) (Stratagene) were addedto 10 μl of a buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM magnesiumchloride and 1 mM DTT, to which 10 units of the restriction enzyme ApaI(Takara Shuzo) were added and reacted at 37° C. for 1 hour. The reactionmixture was ethanol-precipitated, and the precipitate was added to 10 μlof a buffer containing 20 mM Tris-HCl (pH 8.5), 100 mM potassiumchloride, 10 mM magnesium chloride and 1 mM DTT, to which 10 units ofthe restriction enzyme BamHI (Takara Shuzo) were added and reacted at30° C. for 1 hour. The reaction mixture was subjected to agarose gelelectrophoresis to thereby recover about 2 μg of an approx. 3.0 kbApaI-BamHI fragment.

Then, 0.1 μg of the PCR-amplified ApaI-BamHI fragment obtained above and0.1 μg of the ApaI-BamHI fragment from pBluescriptSK(−) obtained abovewere added to sterilized water to give a total volume of 20 μl andligated using Ready-To-Go T4 DNA Ligase (Pharmacia Biotech). Using thethus obtained recombinant plasmid DNA solution, E. coli HB101 wastransformed. From 10 transformant clones, each plasmid DNA was preparedand the base sequence thereof was determined. As a result, plasmidpBShCγ4 shown in FIG. 51 comprising a cDNA of interest coding for humanantibody Cγ4 was obtained.

(2) Construction of an Expression Vector for Anti-Human IL-5R αHumanized Antibodies of Human Antibody IgG4 Subclass

An expression vector for anti-human IL-5R α humanized antibodies ofhuman antibody IgG4 subclass was constructed as described below usingplasmid pBShCγ4 comprising a cDNA coding for human antibody Cγ4 obtainedin subsection (1) of section 7 of Example 2, expression vectorpKANTEX1259 for the anti-human IL-5R α human chimeric antibody KM1399obtained in subsection (1) of section 3 of Example 2 and expressionvector pKANTEX1259HV3LV0 for the anti-human IL-5R α human CDR-graftedantibody KM8399 obtained in subsection (3) of section 6 of Example 2.

Briefly, 4 μg of plasmid pBShCγ4 comprising a cDNA coding for humanantibody Cγ4 were added to 10 μl of a buffer containing 10 mM Tris-HCl(pH 7.5), 10 mM magnesium chloride and 1 mM DTT, to which 10 units ofthe restriction enzyme ApaI (Takara Shuzo) were added and reacted at 37°C. for 1 hour. The reaction mixture was ethanol-precipitated, and theprecipitate was added to 10 μl of a buffer containing 20 mM Tris-HCl (pH8.5), 100 mM potassium chloride, 10 mM magnesium chloride and 1 mM DTT,to which 10 units of the restriction enzyme BamHI (Takara Shuzo) wereadded and reacted at 30° C. for 1 hour. The reaction mixture wassubjected to agarose gel electrophoresis to thereby recover about 1 μgof an approx. 1.0 kb ApaI-BamHI fragment.

Subsequently, 3 μg each of expression vector pKANTEX1259 for theanti-human IL-5R α human type chimeric antibody KM1399 and expressionvector pKANTEX1259HV3LV0 for the anti-human IL-5R α human CDR-graftedantibody KM8399 were added individually to 10 μl of a buffer containing10 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride and 1 mM DTT, to which10 units of the restriction enzyme ApaI (Takara Shuzo) were added andreacted at 37° C. for 1 hour. Both reaction mixtures wereethanol-precipitated, and the precipitates were individually added to 10μl of a buffer containing 20 mM Tris-HCl (pH 8.5), 100 mM potassiumchloride, 10 mM magnesium chloride and 1 mM DTT, to which 10 units ofthe restriction enzyme BamHI (Takara Shuzo) were added and reacted at30° C. for 1 hour. Both reaction mixtures were subjected to agarose gelelectrophoresis to thereby recover about 2 μg of an approx. 12.59 kbApaI-BamHI fragment from each reaction mixture.

A combination of 0.1 μg of the ApaI-BamHI fragment from plasmid pBShCγ4and 0.1 μg of the ApaI-BamHI fragment from plasmid pKANTEX1259 andanother combination of 0.1 μg of the ApaI-BamHI fragment from plasmidpBShCγ4 and 0.1 μg of the ApaI-BamHI fragment from plasmidpKANTEX1259HV3LV0 were added individually to sterilized water to give atotal volume of 20 μl and ligated using Ready-To-Go T4 DNA Ligase(Pharmacia Biotech). Using each of the thus obtained recombinant plasmidDNA solutions, E. coli HB101 was transformed to thereby obtainexpression vector pKANTEX1259γ4 for an anti-human IL-5R α human chimericantibody of IgG4 subclass and expression vector pKANTEX1259HV3LV0γ4 foran anti-human IL-5R α human CDR-grafted antibody of IgG4 subclass shownin FIG. 52.

(3) Expression of Anti-Human IL-5R α Humanized Antibodies in Rat MyelomaYB2/0 Cells (ATCC CRL1581)

The expression of anti-human IL-5R α humanized antibodies in YB2/0 Cellswas performed by the method described in subsection (2) of section 3 ofExample 2 using the expression vector pKANTEX1259γ4 for an anti-humanIL-5R α human chimeric antibody of IgG4 subclass and the expressionvector pKANTEX1259HV3LV0γ4 for an anti-human IL-5R α human CDR-graftedantibody of IgG4 subclass obtained in subsection (2) of section 7 whichwere obtained in Example 2.

As a result, as a transformant producing an anti-human IL-5R α humanchimeric antibody of IgG4 subclass, KM7399 (FERM BP-5649) was obtainedand the anti-human IL-5R α human chimeric antibody of IgG4 subclassproduced by this strain was designated as KM7399. The transformantKM7399 producing the anti-human IL-5R α human chimeric antibody KM7399was deposited with the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology on Sep. 3,1996 under accession number FERM BP-5649. The productivity of theanti-human IL-5R α human chimeric antibody KM7399 in the transformantKM7399 was about 3 μg/10⁶ cells/24 hr.

Also, as a transformant producing an anti-human IL-5R α humanCDR-grafted antibody of IgG4 subclass, KM9399 (FERM BP-5647) wasobtained and the anti-human IL-5R α human CDR-grafted antibody of IgG4subclass produced by this strain was designated as KM9399. Theproductivity of the anti-human IL-5R α human CDR-grafted antibody KM9399in the transformant KM9399 was about 7 μg/10⁶ cells/24 hr. Thetransformant KM9399 producing the anti-human IL-5R α human CDR-graftedantibody KM9399 was deposited with the National Institute of Bioscienceand Human-Technology, Agency of Industrial Science and Technology onSep. 3, 1996 under accession number FERM BP-5647.

(4) Purification of the Anti-Human IL-5R α Humanized Antibodies of HumanAntibody IgG4 Subclass from Culture Supernatants

The transformant KM7399 producing the anti-human IL-5R α human chimericantibody of IgG4 subclass and the transformant KM9399 producing theanti-human IL-5R α human CDR-grafted antibody of IgG4 subclass whichwere obtained in subsection (3) of section 7 of Example 2 were culturedand purified according to the methods described in subsection (3) ofsection 3 of Example 2, to thereby obtain about 1 mg of KM7399 and about5 mg of KM9399. About 4 μg each of the purified anti-human IL-5R αhumanized antibodies of IgG4 subclass KM7399 and KM9399 wereelectrophoresed according to the method described in subsection (3) ofsection 3 of Example 2 to examine their molecular weights. The resultsare shown in FIG. 53. As shown in FIG. 53, the molecular weight of the Hchain of each antibody is about 50 kDa and that of the L chain of eachantibody about 25 kDa under reducing conditions. Thus, the expression ofH chains and L chains of the correct molecular weight was confirmed.Under non-reducing conditions, the molecular weight of each anti-humanIL-5R α humanized antibody is about 140 kDa. Thus, the expression of ahuman CDR-grafted antibody of the correct size composed of two H chainsand two L chains was confirmed. Further, the N terminal amino acidsequences for the H and L chains of the purified anti-human IL-5R αhumanized antibodies of IgG4 subclass KM7399 and KM9399 were analyzedwith a protein sequencer (470A, Applied Biosystems) by the automaticEdman method. As a result, the correct amino acid sequences as expectedwere obtained.

(5) Reactivities of the Anti-Human IL-5R α Humanized Antibodies of HumanAntibody IgG4 Subclass with Human IL-5R α (ELISA method 2)

The reactivities of the anti-human IL-5R α human chimeric antibody ofhuman antibody IgG1 subclass KM1399, the anti-human IL-5R α humanCDR-grafted antibody of human antibody IgG1 subclass KM8399, theanti-human IL-5R α human chimeric antibody of IgG4 subclass KM7399 andthe anti-human IL-5R α human CDR-grafted antibody of IgG4 subclassKM9399 with human IL-5R α were determined by the ELISA method 2described in subsection (2) of section 3 of Example 2. The results areshown in FIG. 54. As shown in FIG. 54, the anti-human IL-5R α humanizedantibodies of human antibody IgG4 subclass proved to have a reactivitywith human IL-5R α as strong as the reactivity of the anti-human IL-5R αhumanized antibodies of IgG1 subclass.

Example 3

1. Confirmation of the Specificity of Anti-hIL-5R α Antibodies

The specificity of anti-hIL-5R α monoclonal antibodies and anti-hIL-5R αhumanized antibodies was confirmed by the following procedures usingimmunocyte staining.

Briefly, 5×10⁵ cells obtained by transfecting a human IL-5R gene intoCTLL-2 cells (ATCC TIB 214) [hereinafter referred to as “CTLL-2(h5R)”][J. Exp. Med., 177, 1523 (1993)] or 5×10⁵ CTLL-2 cells as a control weresuspended in an immunocyte staining buffer (PBS containing 1% BSA, 0.02%EDTA and 0.05% sodium azide) and dispensed into a round bottom 96-wellplate (100 μl/well). After centrifuging at 350×g for 1 minute at 4° C.,the supernatant was discarded. Then, 50 μl of the immunocyte stainingbuffer containing 10 μg/ml of an hIL-5R α antibody were added to eachwell and reacted at 4° C. for 30 minutes. After the reaction, theimmunocyte staining buffer was added (200 μl/well) and centrifuged at350×g for 1 minute at 4° C. and then the supernatant was removed to washthe cells. The washing operation was further repeated twice. Thereafter,50 μl of the immunocyte staining buffer containing FITC-labeledanti-mouse immunoglobulin antibody or FITC-labeled anti-humanimmunoglobulin antibody (both manufactured by Wako Pure ChemicalIndustries, Ltd.) diluted 30 folds with a staining buffer were added toeach well and reacted at 4° C. for 30 minutes. After the reaction, asimilar washing operation was repeated three times. Then, the cells wereanalyzed with a flow cytometer (Coulter).

The results are shown in FIG. 55. Monoclonal antibodies KM1257, KM1259and KM1486 and humanized antibodies KM1399, KM7399, KM8399 and KM9399did not react with CTLL-2 cells, but specifically reacted withCTLL-2(h5R). Thus, it has become clear that humanized antibodies KM1399,KM7399, KM8399 and KM9399 specifically recognize hIL-5R α.

2. Action of Anti-IL-5R α Antibodies to Inhibit the Biological Activityof IL-5

Since CTLL-2(h5R) cells exhibit a proliferation response depending onhuman IL-5 [J. Exp. Med., 177, 1523 (1993)], the effect of theanti-IL-5R α antibodies upon human IL-5 dependent cell proliferation inCTLL-2(h5R) cells was examined. Cell proliferation was evaluated by acolor development method using Cell Counting Kit (Dojin ChemicalLaboratory).

Briefly, 1×10⁴ CTLL-2(h5R) cells were suspended in 50 μl of a normalmedium and dispensed into a 96-well cell culture plate. These cells weremixed with 25 μl/well of various anti-IL-5R α antibodies diluted with anormal medium at 40 μg/ml and with 25 μl/well of a normal mediumcontaining human IL-5 at 0.4 ng/ml as prepared by the method describedin section 3 of Example 1 and cultured in a CO₂ incubator at 37° C. for44 hours. Then, 10 μl/well of Cell Counting Kit solution were added tothe plate and cells were cultured under 5% CO₂ incubator at 37° C. foranother 4 hours. After completion of the cultivation, the absorbance at450 nm was measured with Microwell Plate Reader Emax (Molecular Device).The CTLL-2(h5R) cell proliferation inhibiting activity of each antibodywas calculated by the following formula.

${{Percent}\mspace{14mu}{proliferation}\mspace{14mu}{inhibition}\mspace{14mu}(\%)} = {100 - {\frac{A - C}{B - C} \times 100}}$wherein

-   -   A: OD value in the presence of an antibody    -   B: OD value in the absence of an antibody    -   C: OD value in the absence of human IL-5.

The results are shown in FIG. 56. Monoclonal antibodies KM1259 andKM1486 and humanized antibodies KM1399, KM7399, KM8399 and KM9399inhibited the human IL-5 dependent proliferation of CTLL-2(h5R) cells.However, such activity was not recognized in monoclonal antibody KM1257.

3. Immunocyte Staining of Human Eosinophils

A polymorphonuclear leukocyte fraction was prepared from normal humanblood and cultured for 3 days in the presence of human IL-5 toconcentrate eosinophils. Then, the reactivity of anti-hIL-5R αmonoclonal antibodies was examined with a flow cytometer.

Briefly, polymorphprep (Nicomed) was dispensed into eight 15-mlpolypropylene centrifuge tubes (4 ml/tube) and each plated with 6 ml ofheparinized normal human blood. Then, the tubes were centrifuged at500×g for 30 minutes at room temperature to separate and recoverpolymorphonuclear leukocytes. The polymorphonuclear leukocytes weresuspended in a normal medium to give a concentration of 1.25×10⁷cells/10 ml and dispensed into 4 cell culture dishes in 10 ml portions.Then, human IL-5 was added to the cell suspension at a finalconcentration of 2 ng/ml and the cells were cultured in a CO₂ incubatorat 37° C. for 3 days. After completion of the cultivation, the cellswere centrifuged (1,200 rpm, 5 min.) and suspended in the immunocytestaining buffer to give a concentration of 5×10⁶ cells/ml.

Then, 5×10⁵ cells were dispensed into a round bottom 96-well plate.

After the plate was centrifuged at 350×g for 1 minute at 4° C., thesupernatant was removed. Then, 50 μl of 10% normal mouseserum-containing immunocyte staining buffer were added and reacted at 4°C. for 30 minutes. To the buffer, monoclonal antibody KM1259 labeledwith biotin by conventional methods [“KOSO-KOTAI-HO” (Enzyme AntibodyMethod), Gakusai Kikaku Co., 1985] or, as a control, biotin-labeledanti-human granulocyte colony-stimulating factor monoclonal antibodyKM341 [Agr. Biol. Chem., 53, 1095 (1989)] had been added at aconcentration of 10 μg/ml. After the reaction, 200 μl of the immunocytestaining buffer were added to each well and centrifuged at 350×g for 1minutes at 4° C. and then the supernatant was removed and the cells werewashed. The washing operation was further repeated twice. Thereafter,phycoerythrin-labeled streptavidin (Becton Dickinson) diluted 10 foldswith the immunocyte staining buffer was added (50 μl/well) and reactedat 4° C. for 30 minutes. After the reaction, a similar washing operationwas repeated 3 times. Then, analysis was performed with a flow cytometer(Coulter) by forward scattering and 90-scattering for those cells whichwere recognized as polymorphonuclear leukocytes. Also, the same cellswere stained by the May-Grunwald-Giemsa staining method [“SENSHOKUHOU NOSUBETE” (Review of Staining Methods), Ishiyaku Shuppan Co., 1988] andobserved for polymorphonuclear leukocytes. As a result, it was confirmedthat 75% of the cells were eosinophils.

FIG. 57 shows the histogram obtained. Anti-human IL-5R α monoclonalantibody KM1259 exhibited a definite reactivity. Since 75% of the cellsanalyzed proved to be eosinophils, it was confirmed that anti-humanIL-5R α monoclonal antibody KM1259 has a reactivity with humaneosinophils.

4. Survival Inhibition of Human Eosinophils with Anti-IL-5R α Antibodies

Polymorphonuclear leukocyte fractions were prepared from normal humanblood, and the action of anti-IL-5R α antibodies upon the survival ofeosinophils in the presence of human IL-5 was examined.

Briefly, polymorphprep (Nicomed) was dispensed in 4 ml portions intofifteen 15-ml polypropylene centrifuge tubes and each plated with 8 mlof heparinized normal human blood. Then, the tubes were centrifuged at500×g for 30 minutes at room temperature to separate and recoverpolymorphonuclear leukocytes.

Percoll stock solution was prepared by adding 1 volume of sterilized 1.5M NaCl solution to 9 volumes of Percoll solution (Pharmacia). Then, 80%Percoll solution was prepared by adding 2 volumes of physiologicalsaline to 8 volumes of Percoll stock solution, and 60% Percoll solutionwas prepared by adding 4 volumes of physiological saline to 6 volumes ofPercoll stock solution. For the purpose of removing concomitantmonocytes, 5 ml of 60% Percoll solution were dispensed into each of two15 ml polypropylene centrifuge tubes, plated with the previouslyobtained polymorphonuclear leukocytes suspended in RPMI1640 medium andcentrifuged at 500×g for 30 minutes at room temperature to separate andrecover the precipitated polymorphonuclear leukocytes. Further, for thepurpose of removing concomitant erythrocytes, 5 ml of 80% Percollsolution were dispensed into each of two 15-ml polypropylene centrifugetubes, plated with the previously obtained polymorpho-nuclear leukocytessuspended in RPMI1640 medium and centrifuged at 500×g for 30 minutes atroom temperature to separate and recover the polymorphonuclearleukocytes suspended in the Percoll layer.

Subsequently, cells were dispensed into a 48-well cell culture plate at2×10⁶ cells/well and human IL-5 was added at a final concentration of0.1 ng/ml. Further, each of various anti-IL-5R α antibodies was added ata final concentration of 1 μg/ml. For each antibody, 2 wells werecultured and the solution in each well was adjusted to have a finalvolume of 1 ml. The cells were cultured in a CO₂ incubator at 37° C. for3 days. After completion of the cultivation, the total volume of cellsuspension was recovered from each well and centrifuged (3,000 rpm, 1min.) to recover the cells. The thus obtained cells were suspended in100 μl of PBS. Using 50 μl of this suspension, specimens were preparedwith a cell specimen preparing device, Cytospin3 (Shandon). Afterspecimens were stained by the May-Grunwald-Giemsa staining method, 200cells were observed for each specimen and the number of eosinophils wascounted.

The results are shown in FIG. 58. Monoclonal antibodies KM1259 andKM1486 and humanized antibodies KM1399, KM7399, KM8399 and KM9399 wereall found to have an activity to inhibit the eosinophil survival timeprolongation by IL-5. However, such activity was not recognized inmonoclonal antibody KM1257.

5. Detection of shIL-5R α with an Anti-hIL-5R α Antibodies

Anti-human IL-5R α monoclonal antibody KM1257 diluted with PBS to aconcentration of 10 μg/ml was dispensed into a 96-well EIA plate(Greiner) (50 μl/well) and left at 4° C. overnight to allow the antibodyto be adsorbed. After washing, 100 μl/well of PBS containing 1% bovineserum albumin (BSA)(1% BSA-PBS) were added and reaction was performed atroom temperature for 1 hour to block the remaining active groups. Afterdiscarding 1% BSA-PBS, the purified shIL-5R α obtained in subsection (9)of section 1 of Example 1 that had been diluted with 1% BSA-PBS to aconcentration of 1000–0.1 ng/ml was added and reacted at 4° C.overnight. After washing with Tween-PBS, anti-human IL-5R α monoclonalantibody KM1259 labeled with biotin by conventional methods[“KOSO-KOTAI-HO” (Enzyme Antibody Method), Gakusai Kikaku Co., 1985] anddiluted with 1% BSA-PBS to a concentration of 1 μg/ml was added (50μl/well) and reacted at room temperature for 2 hours. After washing withTween-PBS, avidin-labeled peroxidase (Nippon Reizo) diluted 4000 foldswith 1% BSA-PBS was added (50 μl/well) and reacted at room temperaturefor 1 hour. After washing with Tween-PBS, ABTS substrate solution[2,2-azinobis(3-ethylbenzothiazole-6-sulfonic acid)ammonium] was addedto allow color development. Then, the absorbance at OD of 415 nm wasmeasured (NJ2001; Japan Intermed).

The results are shown in FIG. 59. As a result, it has become clear thatshIL-5R α can be measured by using anti-human IL-5R α monoclonalantibody KM1257 and biotin-labeled anti-human IL-5R α monoclonalantibody KM1259.

6. Detection of shIL-5R α by Western Blotting

The shIL-5R α described in subsection (9) of section 1 of Example 1 wasthermally denatured in SDS-PAGE sample buffer containing2-mercaptoethanol or SDS-PAGE sample buffer not containing2-mercaptoethanol. The resultant mixture was electrophoresed on acommercial SDS-PAGE gradient gel (Atto), and then the protein wastransferred to a PVDF membrane (Millipore). The PVDF membrane was soakedin PBS containing 10% BSA and left at 4° C. overnight for blocking.After completion of the blocking, the membrane was washed thoroughlywith 0.05% Tween-containing PBS. Then, the membrane was soaked in aculture supernatant of the hybridoma obtained in section 5 of Example 1at room temperature for 2 hours and washed thoroughly with 0.05%Tween-containing PBS. Further, the PVDF membrane was soaked at roomtemperature for 1 hour in a solution obtained by dilutingperoxidase-labeled anti-mouse immunoglobulin antibody (Wako PureChemical Industries, Ltd.) with 1% BSA-PBS 1000 folds and then washedthoroughly with 0.05% Tween-containing PBS. After the washing solutionwas removed thoroughly, ECL reagent (Amersham) was applied to the PVFDmembrane and reacted for 1 minute. After removing the excessive reagent,the membrane was sandwiched between two plastic films and placed in anX-ray film sensitizing cassette to thereby sensitize the ECL film. Thus,the reactivity of the antibodies were confirmed.

The results are shown in FIG. 60. KM1257 exhibited reactivity, butKM1259 and KM1486 did not.

7. Immunoprecipitation of shIL-5R α

An anti-mouse immunoglobulin antibody (DAKO) diluted with PBS 50 foldswas dispensed into a 96-well EIA plastic plate (200 μl/well) and left at4° C. overnight to allow the antibody to be adsorbed. After washing withPBS, 300 μl/well of 1% BSA-PBS were added and left at room temperaturefor 1 hour to perform blocking. After washing with PBS, 200 μl each of aculture supernatant of KM1257, KM1259 or KM1486 (they are anti-humanIL-5R α monoclonal antibodies obtained in the preceding Examples) wereadded to each well and left at 4° C. overnight to allow the antibody tobe adsorbed. After washing the plate, the shIL-5R α obtained in section1 of Example 1 and diluted with PBS to a concentration of 10 μg/ml wasdispensed into each well in an amount of 50 μl and reacted at 4° C.overnight. After the plate was washed with 0.05% Tween-containing PBS,5×2-mercaptoethanol-free SDS-PAGE sample buffer [0.31 M Tris (pH 6.8),10% SDS, 50% glycerol] or 5×2 -mercaptoethanol-containing SDS-PAGEsample buffer [0.31 M Tris (pH 6.8), 10% SDS, 50% glycerol, 25%2-mercaptoethanol] was added (50 μl/well) and left at room temperaturefor 2 hours while shaking. The reaction mixture was added to 200 μl ofPBS and heated on a heat block. Then, using a commercial SDS-PAGEgradient gel (Atto), 25 μl of the reaction mixture were separated. Aftercompletion of the electrophoresis, the protein was transferred to a PVDFmembrane (Millipore). The PVDF membrane was subjected to Westernblotting analysis according to the method described in section 6 ofExample 3 and using KM1257, to thereby detect shIL-5R α.

The results are shown in FIG. 61. It has become clear that all ofKM1257, KM1259 and KM1486 immunoprecipitate shIL-5R α.

INDUSTRIAL APPLICABILITY

According to the present invention, monoclonal antibodies KM1257, KM1259and KM1486 are provided which specifically bind to human IL-5 receptor αchain that is believed to be specifically expressed on humaneosinophils. Also, humanized antibodies KM1399, KM8399, KM7399 andKM9399 are provided which specifically bind to human IL-5 receptor αchain that is believed to be specifically expressed on human eosinophilsand which can inhibit the biological activity of human IL-5. Theantibodies of the present invention are useful for immunologicaldetection of human eosinophils in immunocyte staining and diagnosis ortreatment of allergic diseases caused by the inhibition of thebiological activity of IL-5. It should be particularly noted that thehumanized antibodies of the invention are lower in antigenicity than themonoclonal antibodies and expected to maintain their effect for a longperiod.

Deposit of Microorganisms

The following microorganisms have been deposited in accordance with theterms of the Budapest Treaty with the National Institute of Bioscienceand Human-Technology Agency of Industrial Science and Technology,International Depository Authority, 1–3, Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, 305 Japan, on the dates indicated:

Microorganism Accession Number Date Hybridoma KM1259 FERM BP-5134 Jun.13, 1995 Hybridoma KM1399 FERM BP-5650 Sep. 3, 1996 Hybridoma KM1257FERM BP-5133 Jun. 13, 1995 Hybridoma KM1486 FERM BP-5651 Sep. 3, 1996Hybridoma KM7399 FERM BP-5649 Sep. 3, 1996 Hybridoma KM8399 FERM BP-5648Sep. 3, 1996 Hybridoma KM9399 FERM BP-5647 Sep. 3, 1996

1. A method for treating chronic bronchial asthma, comprisingadministering to a mammal in need thereof an effective amount of anantibody which recognizes an epitope at positions 1–313 from theN-terminal amino acid of a human interleukin-5 receptor α chain.
 2. Themethod of claim 1, wherein the antibody is selected from monoclonalantibody, humanized antibody, single chain antibody anddisulfide-stabilized antibody.
 3. The method of claim 2, wherein theantibody is a monoclonal antibody.
 4. The method of claim 3, wherein themonoclonal antibody specifically binds to a human interleukin-5 receptora chain on the surface of an immunocyte.
 5. The method of claim 3,wherein the monoclonal antibody inhibits the eosinophil survival timeprolongation by human interleukin-5.
 6. The method of claim 5, whereinthe monoclonal antibody belongs to IgG1 subclass, CDR sequences in the Vregion of the H chain of the antibody comprise the following amino acidsequences: CDR1: Ser Tyr Val Ile His (Seq ID No. 40); CDR2: Tyr Ile AsnPro Tyr Asn Asp Gly Thr Lys Tyr       Asn Glu Arg Phe Lys Gly (Seq IDNo: 41); and CDR3: Glu Gly Ile Arg Tyr Tyr Gly Leu Leu Gly Asp       Tyr(Seq ID No: 42);

and CDR sequences in the V region of the L chain comprise the followingamino acid sequences: CDR1: Gly Thr Ser Glu Asp Ili Ile Asn Tyr Leu Asn      (Seq ID No: 43); CDR2: His Thr Ser Arg Leu Gln Ser (Seq ID No:44); and CDR3: Gln Gln Gly Tyr Thr Leu Pro Tyr Thr       (Seq ID No:45).


7. The method of claim 6, wherein the monoclonal antibody is monoclonalantibody KM1259 produced by hybridoma KM1259 (FERM BP-5134).
 8. Themethod of claim 5, wherein the monoclonal antibody belongs to IgG1subclass, CDR sequences in the V region of the H chain of the antibodycomprise the following amino acid sequences: CDR1: Asp Thr Tyr Met His(Seq ID No: 46); CDR2: Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Ser      Asp Pro Lys Phe Gln Ala (Seq ID No. 47); and CDR3: Gly Leu Arg LeuArg Phe Phe Asp Tyr       (Seq ID No: 48); and

CDR sequences in the V region of the L chain comprise the followingamino acid sequences: CDR1: Ser Ala Ser Ser Ser Val Ser Tyr Met His      (Seq ID No: 49); CDR2: Asp Thr Ser Lys Leu Ala Ser (Seq ID No:50); and CDR3: Gln Gln Trp Ser Ser Asn Pro Pro Ile Thr       (Seq ID No:51).


9. The meted of claim 8, wherein the monoclonal antibody is monoclonalantibody KM1486 produced by hybridoma KM1486 (FERM BP-5651).
 10. Themethod of claim 2, wherein the antibody is a humanized antibody.
 11. Themethod of claim 10, wherein the humanized antibody reacts specificallywith the human interleukin-5-receptor α chain by immunocyte staining.12. The method of claim 10, wherein the humanized antibody inhibits theeosinophil survival time prolongation by human interleukin-5.
 13. Themethod of claim 11 or 12, wherein the antibody belongs to human antibodyIgG class.
 14. The method of claim 10, wherein the humanized antibody isa human chimeric antibody.
 15. The method of claim 14, wherein the humanchimeric antibody is a chimeric antibody comprising the V region of theH chain and the V region of the L chain of a non-human animal antibody,as well as the constant region (C region) of the H chain and the Cregion of the L chain of a human antibody.
 16. The method of claim 15,wherein the V region of the H chain of the antibody comprises the aminoacid sequence of SEQ ID NO: 27 and the V region of the L chain of theantibody comprises the amino acid sequence of SEQ ID NO:
 29. 17. Themethod of claim 15, wherein the V region of the H chain of the antibodycomprises the amino acid sequence of SEQ ID NO: 31 and the V region ofthe L chain of the antibody comprises the amino acid sequence of SEQ IDNO:
 33. 18. The method of claim 15, wherein the antibody is antibodyKM1399 wherein the C region of the H chain of the antibody is in humanantibody IgG1 subclass.
 19. The method of claim 15, wherein the antibodyis antibody KM7399 wherein the C region of the H chain of the antibodyis in human antibody IgG4 subclass.
 20. The method of claim 10, whereinthe humanized antibody is a human CDR-grafted antibody.
 21. The methodof claim 20, wherein the human CDR-grafted antibody is obtained byreplacing CDR sequences in the V region of the H chain and the V regionof the L chain of a human antibody with CDR sequences in the V region ofthe H chain and the V region of the L chain of a non-human animalantibody.
 22. The method of claim 21, wherein CDR sequences in the Vregion of the H chain of the antibody comprises CDR sequences in the Vregion of the H chain of the antibody of claim 6 and CDR sequences inthe V region of the L chain of the antibody comprises CDR sequences inthe V region of the L chain of the antibody of claim
 6. 23. The methodof claim 21, wherein CDR sequences in the V region of the H chain of theantibody comprises CDR sequences in the V region of the H chain of theantibody of claim 8 and CDR sequences in the V region of the L chain ofthe antibody comprises CDR sequences in the V region of the L chain ofthe antibody of claim
 8. 24. The method of claim 22, wherein theantibody is antibody KM8399 wherein the C region of the H chain of theantibody belongs to human antibody IgG1 subclass.
 25. The method ofclaim 22, wherein the antibody is antibody KM9399 wherein the C regionof the H chain of the antibody is in a human antibody IgG4 subclass. 26.The method of claim 2, wherein the antibody is a single chain antibody.27. The method of claim 26, wherein the single chain antibody inhibitsthe eosinophil survival time prolongation by human interleukin-5. 28.The method of claim 26, wherein the single chain antibody comprises theV region of the H chain and the V region of the L chain of a humanizedantibody.
 29. The method of claim 26, wherein CDR sequences in the Vregion of the H chain and the V region of the L chain of the singlechain antibody comprise CDR sequences in the V region of the H chain andthe V region of the L chain of the monoclonal antibody of claim
 6. 30.The method of claim 26, wherein CDR sequences in the V region of the Hchain and the V region of the L chain of the single chain antibodycomprise CDR sequences in the V region of the H chain and the V regionof the L chain of the monoclonal antibody of claim
 8. 31. The method ofclaim 2, wherein the antibody is a disulfide-stabilized antibody. 32.The method of claim 31, wherein the disulfide-stabilized antibodyinhibits the eosinophil survival time prolongation by humaninterleukin-5.
 33. The method of claim 32, wherein thedisulfide-stabilized antibody comprises the V region of the H chain andthe V region of the L chain of a humanized antibody.
 34. The method ofclaim 32, wherein CDR sequences in the V region of the H chain and the Vregion of the L chain of the disulfide-stabilized antibody comprise CDRsequences in the V region of the H chain and the V region of the L chainof the monoclonal antibody of claim
 6. 35. The method of claim 32,wherein CDR sequences in the V region of the H chain and the V region ofthe L chain of the single chain antibody comprise CDR sequences in the Vregion of the H chain and the V region of the L chain of the monoclonalantibody of claim 8.